Immunogenic combinations

ABSTRACT

This disclosure provides immunogenic combinations that include a) an immunogenic component containing a peptide or polypeptide antigen of a respiratory pathogen; and b) an immunogenic component containing a nucleic acid encoding an antigen of the same respiratory pathogen, wherein the immunogenic components are formulated for concurrent, e.g., co-localized, administration. More specifically, the respiratory pathogen is respiratory syncytial virus (RSV). This disclosure also concerns provides the use of such immunogenic combinations, and methods for administering such immunogenic combinations to elicit an immune response specific for the respiratory pathogen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of copending application Ser. No.15/318,490, filed on Dec. 13, 2016, which is the National Phase under 35U.S.C. § 371 of International Application No. PCT/EP2015/063248, filedon Jun. 12, 2015, which claims the benefit under 35 U.S.C. § 119(e) toU.S. Provisional Application No. 62/011,712, filed on Jun. 13, 2014, allof which are hereby expressly incorporated by reference into the presentapplication.

REFERENCE TO ELECTRONIC SEQUENCE LISTING

The application contains a Sequence Listing which has been submittedelectronically in .XML format and is hereby incorporated by reference inits entirety. Said .XML copy, created on Dec. 14, 2022, is named“2022-12-14_SequenceListing_2801-0347PUS2.xml” and is 11,019 bytes insize. The sequence listing contained in this .XML file is part of thespecification and is hereby incorporated by reference herein in itsentirety.

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure, as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

BACKGROUND

This disclosure concerns the field of immunology. More particularly thisdisclosure relates to a method for eliciting an immune response toprotect against respiratory pathogens.

Because the respiratory tract is in direct contact with the environmentand is exposed to numerous airborne organisms, the respiratory tract isthe most frequent site of infection by pathogenic organisms. Suchinfections can result in symptoms and disease ranging from the commoncold, to bronchitis, bronchiolitis and pneumonia, as well as severe andchronic conditions. Common causal agents of respiratory infectioninclude both viruses, such as rhinoviruses, coronaviruses, influenzavirus, respiratory syncytial virus and other paramyxoviruses,adenovirus, and bacteria, such as Streptococcus sp., Corynebacteriumdiptheriae, Bordatella pertussis, Haemophilus influenza, andMycobacterium tuberculosis.

Effective vaccines have been produced to protect children and adultsfrom many of these respiratory pathogens. However, developing vaccinesthat are effective against other respiratory pathogens has provenchallenging. Accordingly, new strategies for safe and effective vaccinesthat are effective against respiratory pathogens are necessary,especially to protect the very young, the elderly and other vulnerableindividuals.

BRIEF SUMMARY

This disclosure concerns an immunogenic combination that includes a) animmunogenic component containing a peptide or polypeptide antigen of arespiratory pathogen; and b) an immunogenic component containing anucleic acid encoding an antigen of the same respiratory pathogen,wherein the immunogenic components are formulated for concurrent, e.g.,co-localized, administration. More specifically, the respiratorypathogen is respiratory syncytial virus (RSV). This disclosure alsoconcerns the use of such immunogenic combinations, and methods foradministering such immunogenic combinations to elicit an immune responsespecific for the respiratory pathogen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating RSV A neutralizing titre followingimmunization according to various regimens.

FIG. 2 is a graph illustrating interferon-gamma (IFNγ) productionfollowing immunization according to various regimens.

FIG. 3 is a graph illustrating RSV A neutralizing titre followingimmunization according to various regimens.

FIG. 4 is a graph illustrating RSV M2-1-specific CD8+ T cells followingimmunization according to various regimens.

FIG. 5 is a graph illustrating neutralizing titre following immunizationaccording to various regimens.

FIG. 6 is a graph illustrating RSV titre in lung following immunizationaccording to various regimens.

FIG. 7 is a graph illustrating the ratio of Th1 and Th2 T cellsfollowing immunization according to various regimens.

FIG. 8 is a graph illustrating the number of mucus-producing cells inlungs following challenge of subjects immunized according to variousregimens.

FIG. 9 is a graph illustrating the number of eosinophils inbronchoalveolar lavage (BAL) fluid following challenge of subjectsimmunized according to various regimens.

FIG. 10 is a graph illustrating RSV A neutralizing titre followingimmunization according to various regimens.

FIG. 11 is a graph illustrating RSV M2-1-specific CD8+ T cells followingimmunization according to various regimens.

FIG. 12 is a graph illustrating lung viral titre following challenge ofsubjects immunized according to various regimens.

FIG. 13 is a graph illustrating the ratio of Th1 and Th2 T cellsfollowing immunization according to various regimens.

FIG. 14 is a graph illustrating RSV M2-1-specific CD8+ T cells followingchallenge of subjects immunized according to various regimens.

FIG. 15 is a graph illustrating the number of mucus-producing cells inlungs following challenge of subjects immunized according to variousregimens.

DETAILED DESCRIPTION Introduction

Developing safe and effective vaccines against certain respiratorypathogens has proven particularly challenging. The present disclosureconcerns improved compositions and methods with increased immunogenicefficacy. More specifically, this disclosure provides immunogeniccombinations for concurrent, and preferably co-localized administration,that elicit potent B and T cell responses, thereby enhancingimmunogenicity, safety, and ultimately protection, against respiratorypathogens.

One aspect of the disclosure relates to an immunogenic combinationcomprising:

-   -   a) at least a first immunogenic component comprising a peptide        or polypeptide antigen of a respiratory pathogen; and b) at        least a second immunogenic component comprising a nucleic acid        encoding an antigen of the same respiratory pathogen; in which        the first immunogenic component and the second immunogenic        component are formulated for concurrent administration.

In certain embodiments, the respiratory pathogen is a virus, such as aparamyxovirus. In one specific embodiment, the respiratory pathogen isRespiratory Syncytial Virus (RSV). In such an embodiment, the antigenscan be selected from RSV antigens including the fusion protein (F), theattachment protein (G), the matrix protein (M2—which may include eitheror both of the M2-1 (which may be written herein as M2.1) and M2-2 geneproducts) and the nucleoprotein (N). In a specific embodiment, thepolypeptide antigen component contains a F protein antigen that isconformationally constrained in either a pre-fusion or a post-fusionconformation.

In certain embodiments of the immunogenic combination, the antigen ofthe first component and the antigen encoded by the nucleic acid of thesecond component share substantial sequence identity, such as about 70%sequence. In an exemplary embodiment, the immunogenic combinationincludes a first component that includes a polypeptide with the aminoacid sequence represented by; and/or a second component that includes anucleic acid that encodes a polypeptide with the amino acid sequencerepresented by:

-   -   a) a polypeptide comprising SEQ ID NO:2;    -   b) a polypeptide with at least 80% sequence identity to SEQ ID        NO:2, which polypeptide comprises an amino acid sequence        corresponding to the RSV F protein polypeptide of a naturally        occurring RSV strain; or    -   c) a polypeptide with at least 80% sequence identity to SEQ ID        NO:2, which polypeptide comprises an amino acid sequence that        does not correspond to a naturally occurring RSV strain.

In certain embodiments, the first immunogenic component contains andsecond immunogenic component encodes homologous antigens. Suchhomologous antigens may either be identical in sequence or non-identicalin sequence. For example, the antigens can possess partially identicalamino acid sequences. Favorably, such antigens include one or moreidentical or overlapping immunogenic epitopes.

For example, one exemplary immunogenic combination for eliciting animmune response specific for RSV comprises a first immunogenic componentthat contains a polypeptide of at least about 500 amino acids of an RSVF protein, the second immunogenic component contains a nucleic acid thatencodes an identical or non-identical polypeptide (e.g., of at leastabout 500 amino acids of an RSV F protein). When non-identical, the RSVF protein polypeptides possess at least 80% sequence identity within theF1 and F2 domains. Favorably, the polypeptide of the first immunogeniccomponent and or the polypeptide encoded by the second immunogeniccomponent include or are an ectodomain of an RSV F Protein (F_(TM)).

In certain embodiments, the first immunogenic component and/or thesecond immunogenic components contain a plurality of antigens (e.g., ofa respiratory pathogen, in particular of RSV).

As described above, the first immunogenic component contains a peptideor polypeptide (or fragment thereof) antigen of a respiratory pathogen.Such a peptide or polypeptide can optionally be in the form of aparticle, such as a VLP or virosome, or a nanoscale biological particle.

Likewise, as described above, the second immunogenic component containsa nucleic acid that encodes an antigen of a respiratory pathogen. Bothdeoxy-ribonucleic acids and ribonucleic acids are suitable. Favorably,the nucleic acid is a nucleic acid other than a plasmid DNA. The nucleicacid can be included in a DNA or RNA vector, such as a replicable vector(e.g., a viral replicon, a self-amplifying nucleic acid), or in a virus(e.g., a live attenuated virus) or viral vector (e.g., replicationproficient or replication deficient viral vector). Suitable viralvectors include but are not limited to an adenovirus, a modifiedvaccinia ankara virus (MVA), a paramyxovirus, a Newcastle disease virus,an alphavirus, a retrovirus, a lentivirus, an adeno-associated virus(AAV), a vesicular stomatitis virus, and a flavivirus. Optionally, theviral vector is replication defective.

In one embodiment, the first component contains an RSV F proteinantigen, and the second component contains a nucleic acid that encodesan RSV F antigen and RSV, M and N antigens. More specifically, the firstcomponent contains an RSV F protein antigen conformationally constrainedin the prefusion conformation, and the second component contains anucleic acid that encodes an RSV FΔTM antigen and RSV M2-1 and Nantigens, wherein a self-cleavage site is included between the RSV FΔTMantigen and the RSV M2-1 and a flexible linker is included between theRSV M2-1 and N antigens.

The first and second immunogenic components of the immunogeniccombination can be formulated (for example, with a pharmaceuticallyacceptable buffer, carrier, excipient and/or adjuvant) in differentcompositions. Alternatively, the first and second immunogenic componentscan be co-formulated in a single composition for administration (eitherat the point of manufacturing, e.g., in a stable co-formulation suitablefor storage, distribution, and administration, or at the point ofdelivery prior to administration). When the first and second immunogeniccomponents are formulated in different compositions, they are favorablyadministered colocationally at or near the same site. For example, thefirst and second immunogenic components can be administered parentallyby injection (e.g., via an administration route selected fromintramuscular, transdermal, intradermal, sub-cutaneous) to the same sideor extremity (co-lateral) administration). Alternatively, the first andsecond immunogenic components can be administered via mucosal,intranasal, oral, sublingual, or aerosol route or delivered to the lungin the form of a powder (particulate) or liquid.

In formulations containing an adjuvant, the adjuvant can include one ormore of a metallic salt (Aluminum hydroxide, Aluminum phosphate,Aluminum potassium sulfate, aluminum hydroxyphosphate sulfate, Calciumhydroxide, Calcium fluoride, Calcium phosphate, Cerium(III) nitratehexahydrate, Zinc sulfate heptahydrate), 3-D-monophosphoryl-lipid-A(MPL), a saponin, an oil and water emulsion, and/or a nanoparticle.

Another aspect of the present disclosure concerns use of the immunogeniccombinations described above in medicine, e.g., for the prevention,reduction or treatment in a subject (such as a human subject, forexample, a neonate, and infant, a child, an adolescent, an adult, e.g.,a pregnant female or an elderly adult) of infection by or diseaseassociated with a respiratory pathogen. Accordingly, also included aremethods for eliciting an immune response specific for a pathogen byadministering the immunogenic combinations described above. Theadministration can be in a vaccination regimen for the prevention,reduction or treatment of infection by or disease associated with arespiratory pathogen, such as a virus or bacterium that causes aninfection of the upper and/or lower respiratory tract and/or the lungs.As disclosed above, in one particular embodiment, the use, method (orvaccination regimen) is for the prevention, reduction or treatment ofinfection by or disease associated with a paramyxovirus, such asRespiratory Syncytial Virus (RSV). In such a method, use or vaccinationregimen, the first and second immunogenic components are administeredconcurrently (at or about the same time), and generally at or near thesame location (e.g., co-laterally when administered by injection). Whenadministered by injection, the first and second immunogenic componentscan be co-formulated, or individually formulated, in which case it iscontemplated that the first and second immunogenic components areadministered using a multi-chamber syringe or by a needle-free device,such as a transdermal patch.

In some embodiments, the method, use or vaccination regimen concurrentlyand/or co-locally administering the first and second immunogeniccomponent elicits an immune response specific for the pathogen that isgreater than the additive effect of an immune response elicited by thefirst immunogenic component and the second immunogenic component whenadministered or used separately. Favorably, administration of theimmunogenic combinations disclosed herein elicits a humoral immuneresponse, a cellular immune response or both a humoral immune responseand a cellular immune response.

Also disclosed are kits containing the immunogenic combination describedherein. Such kits also favorably include at least one device foradministering the immunogenic combination, such as one or morepre-filled syringes, e.g., a multi-chambered syringe or a needle-freedevice, such as a transdermal patch.

Terms

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology can be found in Benjamin Lewin, Genes V,published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrewet al. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. The term “plurality” refers to two or more. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Additionally,numerical limitations given with respect to concentrations or levels ofa substance, such as an antigen, are intended to be approximate. Thus,where a concentration is indicated to be at least (for example) 200 pg,it is intended that the concentration be understood to be at leastapproximately (or “about” or “˜”) 200 pg.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” Thus, unless the context requires otherwise, the word“comprises,” and variations such as “comprise” and “comprising” will beunderstood to imply the inclusion of a stated compound or composition(e.g., nucleic acid, polypeptide, antigen) or step, or group ofcompounds or steps, but not to the exclusion of any other compounds,composition, steps, or groups thereof. The abbreviation, “e.g.” isderived from the Latin exempli gratia, and is used herein to indicate anon-limiting example. Thus, the abbreviation “e.g.” is synonymous withthe term “for example.”

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of terms are provided. Additionalterms and explanations can be provided in the context of thisdisclosure.

The term “immunogenic” when referring, e.g., to a composition, meansthat the composition is capable of eliciting a specific immune response,e.g., against a pathogen, such as a respiratory pathogen. An“immunogenic epitope” is a portion of an antigen to which a specificimmune response (e.g., a B cell response and/or a T cell response) isdirected, for example, via specific binding of a T cell receptor and/orantibody.

An “immunogenic composition” or “immunogenic component” is a compositionsuitable for administration to a human or animal subject (e.g., in anexperimental setting) that is capable of eliciting a specific immuneresponse. As such, an immunogenic composition includes one or moreantigens (for example, a polypeptide antigen or nucleic acid that encodea polypeptide antigen) or antigenic epitopes. An immunogenic compositioncan also include one or more additional components capable of elicitingor enhancing an immune response, such as an excipient, carrier, and/oradjuvant. In certain instances, immunogenic compositions areadministered to elicit an immune response that protects the subjectagainst symptoms or conditions induced by a pathogen. In some cases,symptoms or disease caused by a pathogen is prevented (or reduced orameliorated) by inhibiting replication of the pathogen (e.g.,respiratory pathogen) following exposure of the subject to the pathogen.In the context of this disclosure, the term immunogenic composition willbe understood to encompass compositions that are intended foradministration to a subject or population of subjects for the purpose ofeliciting a protective or palliative immune response against respiratorypathogens (that is, vaccine compositions or vaccines). In the context ofthe present disclosure an “immunogenic component” refers to animmunogenic composition that is preferentially used in combination withone or more additional immunogenic components or compositions. An“immunogenic combination” is an immunogenic composition that comprisesor includes more than one (a plurality of) substituent “immunogeniccomponents”.

An “immune response” is a response of a cell of the immune system, suchas, but not limited to, a B cell, T cell, NK cell, monocyte, dendriticcell, or polymorphonuclear cell to a stimulus. An immune response can bea B cell response, which results in the production of specificantibodies, such as antigen specific neutralizing antibodies. An immuneresponse can also be a T cell response, such as a CD4+ response or aCD8+ response. In some cases, the response is specific for a particularantigen (that is, an “antigen-specific response”). If the antigen isderived from a pathogen, the antigen-specific response is a“pathogen-specific response.” A “protective immune response” is animmune response that inhibits a detrimental function or activity of apathogen, reduces infection by a pathogen, or decreases symptoms(including death) that result from infection by the pathogen. Aprotective immune response can be measured, for example, by theinhibition of viral replication or plaque formation in a plaquereduction assay or by a functional antibody response, by the reductionof signs or symptoms, or by measuring resistance to pathogen challengein vivo.

An “antigen” is a compound, composition, or substance that can stimulatethe production of antibodies and/or a T cell response in an animal,including compositions that are injected, absorbed or otherwiseintroduced into an animal (such as a human being). The term “antigen”includes all related antigenic epitopes. The term “epitope” or“antigenic determinant” refers to a site on an antigen to which B and/orT cells respond. The “dominant antigenic epitopes” or “dominant epitope”are those epitopes to which a functionally significant host immuneresponse, e.g., an antibody response or a T-cell response, is made. Insome cases, the host response to one or more dominant epitopes providesa protective immune response against a pathogen. The term “T-cellepitope” refers to an epitope that when bound to an appropriate MHCmolecule is specifically bound by a T cell (via a T cell receptor). A“B-cell epitope” is an epitope that is specifically bound by an antibody(or B cell receptor molecule).

The term “polypeptide” refers to a polymer in which the monomers areamino acid residues which are joined together through amide bonds. Theterms “polypeptide” or “protein” as used herein are intended toencompass any amino acid sequence and include modified sequences such asglycoproteins. The term “polypeptide” is specifically intended to covernaturally occurring proteins, as well as those which are recombinantlyor synthetically produced. The term “fragment,” in reference to apolypeptide, refers to a portion (that is, a subsequence) of apolypeptide. The term “immunogenic fragment” refers to all fragments ofa polypeptide that retain at least one predominant immunogenic epitopeof the full-length reference protein or polypeptide. The term “peptide”refers to a polymer of amino acids (joined through amide bonds),generally of less than 100 amino acids in length (e.g., of less than 50,or less than 40, or less than 30, or less than 25, or less than 20, orless than 15, or less than 10 amino acids in length). Orientation withina peptide or polypeptide is generally recited in an N-terminal toC-terminal direction, defined by the orientation of the amino andcarboxy moieties of individual amino acids. Polypeptides are translatedfrom the N or amino-terminus towards the C or carboxy-terminus.

The terms “nucleic acid” and “polynucleotide” refer to a polymeric formof nucleotides at least 10 bases in length. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single and double, or positive andnegative, forms of DNA or RNA. By “isolated” nucleic acid (orpolynucleotide) is meant a nucleic acid (or polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.In one embodiment, a polynucleotide encodes a polypeptide. The 5′ and 3′direction of a nucleic acid is defined by reference to the connectivityof individual nucleotide units, and designated in accordance with thecarbon positions of the deoxyribose (or ribose) sugar ring. Theinformational (coding) content of a nucleic acid sequence is read in a5′ to 3′ direction.

In the context of a nucleic acid, the term “vector” refers to a nucleicacid that is capable of incorporating or carrying a nucleic acidobtained from a different source (an “insert”) and replicating and/orexpressing the inserted polynucleotide sequence, when introduced into acell (e.g., a host cell). A nucleic acid vector can be either DNA orRNA. The term vector will be understood to include, e.g., plasmids,cosmids, phage, virus vectors, autonomously replicating viral nucleicacids, replicons, artificial chromosomes, and the like.

An “adjuvant” is an agent that enhances the production of an immuneresponse in a non-specific manner. Common adjuvants include suspensionsof minerals (alum, aluminum hydroxide, aluminum phosphate) onto whichantigen is adsorbed; emulsions, including water-in-oil, and oil-in-water(and variants thereof, including double emulsions and reversibleemulsions), liposaccharides, lipopolysaccharides, immunostimulatorynucleic acids (such as CpG oligonucleotides), liposomes, Toll-likeReceptor agonists (particularly, TLR2, TLR4, TLR7/8 and TLR9 agonists),and various combinations of such components.

A “vaccination regimen” refers to a protocol (e.g., a sequence) ofadministrations of an immunogenic composition or combination ofimmunogenic compositions determined to elicit a desired immune responsespecific for an antigen (or plurality of antigens).

The term “concurrent” or “concurrently” means at or about the same time.Concurrent administration of an immunogenic combination meansadministration of two or more immunogenic components at or about thesame time. In the context of a vaccination regimen, the term shall beunderstood to mean administration of two or more immunogenic componentsto elicit a kinetically associated immune response (e.g., a primaryresponse, or a response that restimulates or boosts a secondary ormemory response). Typically, concurrent administration with two or moreimmunogenic components (e.g., of an immunogenic combination) occursbetween 0 and 10 days. Typically, concurrent administration occurs nolonger than about 7 days apart, such as about 5 days, preferably nolater than about 3 days, such as within 24 hours, such as within about 8hours or less. Commonly, concurrent administration of two or moreimmunogenic components occurs within about 2 hours or less, such thatthe first and at least second immunogenic composition or component areadministered within a period of 2 hours, a period of 1 hour, or withinabout 30 minutes, or about 10 minutes. In some instances, concurrentadministration is performed at the same time, e.g., in one or moreinjections.

The term “co-administration” in relation to the administration to asubject of more than one immunogenic composition means administration ofthe one immunogenic compositions concurrently.

The term “colocationally” means that two compositions (for example,immunogenic compositions or immunogenic components of an immunogeniccombination) are administered to the same (or about the same) locationon the body of the recipient subject. The same location will beunderstood herein to mean to the same or approximately the same site ororifice. For example, in the case of mucosal administration, animmunogenic combination can be administered to the same orifice (e.g.,the mouth or nose). In the case of parenteral administration,colocationally means in proximity at the same (or approximately thesame) site on the body, such as to the same site (e.g., by the samedevice), or within about 10 cm, or more commonly within about 5 cm, suchas within about 2 cm, or within 1 cm. In some instances, the two or morecomponents are combined (co-formulated) in a single composition foradministration to the same site. The term “co-lateral” or “co-laterally”in the case of parenteral administration means to the same side of thebody in a grossly bilaterally symmetrical organism (such as a mammal,for example, a human or animal subject). The term co-lateral iscontrasted with the term “contra-lateral”, which refers to oppositesides of a grossly bilaterally symmetrical organism.

A “subject” is a living multi-cellular vertebrate organism. In thecontext of this disclosure, the subject can be an experimental subject,such as a non-human animal, e.g., a mouse, a cotton rat, or a non-humanprimate. Alternatively, the subject can be a human subject.

Immunogenic Combinations

This disclosure relates to immunogenic combinations capable of elicitingstrong T-cell and B-cell responses, e.g., in naïve subjects. Theimmunogenic combinations disclosed herein contain: a) at least a firstimmunogenic component comprising a peptide or polypeptide antigen; andb) at least a second immunogenic component comprising a nucleic acidencoding an antigen. The immunogenic components containing the peptideor polypeptide antigen and the nucleic acid that encodes an antigen areformulated for concurrent administration to a recipient. Whenconcurrently administered, the first and second immunogenic componentselicit a stronger or broader (e.g., more diverse and/or qualitativelymore balanced) immune response than the additive response of eachcomponent when administered separately.

Although protective immune responses may be characterized as beingpredominantly either B cell (antibodies, for example, neutralizingantibodies), or T cell responses (e.g., a CD8+ T cell response), in someinstances it is both desirable and advantageous to generate strong Bcell and strong T cell responses specific for antigens (either the sameor different) of a pathogen. Unfortunately, many vaccination regimensdisproportionately elicit either a B cell or a T cell response without aprotective increase in complementary response. This failure to elicitboth B and T cell responses can be particularly important in protectingyoung infants, both due to the relative immaturity of the infant'simmune system, and also due to the presence of maternal antibodies.

Typical vaccine regimens involve the repeated administration of anidentical immunogenic composition (e.g., a vaccine). The firstadministration (designated for convenience a priming administration or“prime”) induces proliferation and maturation of B and/or T cellprecursors specific to one or more immunogenic epitopes present on theantigen (induction phase). The second (and in some cases subsequent)administration (designated for convenience a boosting administration or“boost”), further stimulates and potentially selects an anamnesticresponse of cells elicited by the prior administration(s). Thus, a biastowards either a B cell or a T cell immune response is amplified bysubsequent administrations of the same immunogenic composition. Incertain instances, the first administration is to a naïve subject.

The present invention is predicated on the demonstration that concurrentadministration of a protein (or peptide) antigen, as evidenced bypathogen specific neutralizing antibodies, with a nucleic acid thatencodes an antigen of the same pathogen, is capable of eliciting B and Tcell responses specific for the antigen and which exceed (bothquantitatively and qualitatively) the cumulative response elicited byadministering the two compositions separately and/or subsequently.Moreover, this combination can be achieved without immune interference,which is frequently observed with the combination of multiple (e.g.,related) antigens. In some cases, immune interference diminishes theelicited immune response so significantly as to render combinationvaccination counterproductive. Immune interference has previously beenreported not only in the context of vaccines (e.g., combinationvaccines) for respiratory pathogens, but also in the context ofinterference between maternally derived antibodies and vaccination ofinfants early in life (e.g., during the neonatal period throughapproximately 6 months of age).

As used above, “concurrent” or “simultaneous” administration refers tothe same ongoing immune response. Preferably both compositions areadministered at the same time (concurrent administration of bothDNA+protein), however, one compound could be administered within a fewminutes (for example, at the same medical appointment or doctor'svisit), within a few hours, or within a few week's time (preferably 0-10days) of the other (initial) administration and still be considered as“concurrent” since they both act during the same ongoing immuneresponse.

Normally, when a polypeptide is administered, the immune response isconsidered immediate in that an immune response will initiate as soon asthe antigen is exposed to the immune system. In contrast, when nucleicacid is administered, peak antigen expression (in vivo) is observed 3-7days after administration, and thus antigen exposure to the immunesystem may be considered “delayed” when compared to the kinetics ofprotein vaccination. Regardless of this difference in kinetics,co-administration of nucleic acid and polypeptide can be considered“concurrent” by understanding that they are both functionally presentduring the process of an ongoing immune response. In order to presentboth immunogenic components (that is both as a polypeptide as such andpolypeptide expressed by the administered nucleic acid), virtuallysimultaneously to the immune system, formulations can be conceivedwherein the polypeptide is contained in such a way that its release fromthe formulation is delayed after the administration. This allows theexpression of polypeptide from the polynucleotide to occur first, whichis then subsequently complemented by the delayed released polypeptidefrom the formulation.

In one particular embodiment of the immunogenic combination disclosedherein involves the concurrent administration of both nucleic acid andprotein where the protein (polypeptide) is present or administered inthe form of delayed-release particles intended to hide the antigen fromthe immune system for a short period of time. Preferably such period isbetween 0-10 days. Typically, concurrent administration occurs at aninterval of no greater than about 7 days apart, such as about 5 days,and more typically in an interval of no longer than about 3 days, suchas within 24 hours, such as within about 8 hours or less. Commonly,concurrent administration of two or more immunogenic components occurswithin about 2 hours or less, such that the first and at least secondimmunogenic composition or component are administered within a period of2 hours, a period of 1 hour, or within about 30 minutes, or within about20 minutes or about 10 minutes, or within about 5 minutes, or withinabout 2 minutes. In some instances, concurrent administration isperformed at the same time, e.g., in one or more injections. It isenvisioned that concurrent administration can be performed convenientlyat a single medical appointment or doctor's visit.

Regardless of the different modes or possibilities of concurrent orsimultaneous administration, as described above, it is important thatboth immunogenic components are present during the induction phase of anongoing immune response. In comparison to this, the prime boost conceptrefers to 2 separate immune responses: (i) an initial priming of theimmune system with a polynucleotide followed by (ii) a secondary orboosting of the immune system with a polypeptide many weeks or monthsafter the primary immune response has been established.

The nucleic acid and protein components can thus be administered as twoseparate events or combined (admixed) to permit one administration.Preferably, the nucleic acid and protein components are admixed.Admixing can occur just prior to use, or when the two components aremanufactured (and formulated), or any time in between.

The disclosed immunogenic combinations have been demonstrated tocircumvent immune interference and exhibit superior or broader (e.g.,more diverse and/or balanced) immunogenicity as compared to separateadministration of either protein or nucleic acid based vaccines. Thedisclosed immunogenic combinations are particularly suitable foreliciting an immune response against a respiratory pathogen,particularly against RSV.

A respiratory pathogen is understood to mean a pathogen that infectscells of the upper (nasal cavity, pharynx and larynx) and/or lower(trachea, bronchi, lungs) respiratory tract, in severe cases causingbronchitis, bronchiolitis and/or pneumonia. Respiratory pathogensinclude both bacteria and viruses. Bacterial pathogens that infect therespiratory tract include, for example, S. pneumoniae, N. meningitide,as well as, H. influenzae and M. catarrhalis, which are involved incommunity acquired pneumonia (CAP) and acute exacerbation of chronicbronchitis (AECB). C. diptheriae is the causative agent of the upperrespiratory tract infection, Diphtheria, while B. pertussis causeswhooping cough or Pertussis. Other bacteria that cause respiratory tractinfections include: C. pneumoniae, M. pneumoniae and L. pneumophila.Also included among the bacterial respiratory pathogens is M.tuberculosis, which causes (among other manifestation) pulmonarytuberculosis, B. anthracis, which when inhaled causes lethal respiratorytract infections, and Y. pestis, which is capable of causing pneumonicplague.

A broad range of viruses also infect and cause disease of therespiratory tract. Most predominantly, viral respiratory pathogensinclude members of the Orthomyxoviridae, for example, influenza virusand Paramyxoviridae, including Respiratory Syncytial Virus (RSV), themetapneumoviruses (e.g., human metapneumovirus, hMPV), parainfluenzaviruses (PIV), as well as the viruses that cause measles(Morbillivirus), mumps (Rubulavirus), and Newcastle disease.Adenoviruses are also common respiratory pathogens. Although lessfrequent, certain coronaviruses (e.g., SARS virus) can cause severerespiratory disease. In addition, Rubivirus, a Togavirus, which causesRubella, can lead to respiratory tract infections.

Thus, in the context of this disclosure, broadly speaking, bothbacterial respiratory pathogens, and viral respiratory pathogens, aresuitable targets for the immunogenic combinations and methods hereindescribed. In certain embodiments, the respiratory pathogen is selectedto be a bacterium. In an immunogenic combination (as described herein)to prevent or reduce (or to treat) infection or disease caused by abacterial respiratory pathogen, the immunogenic compositions incorporateantigens selected from a bacterium, such as an antigen selected from: S.pneumoniae, N. meningitide, H. influenza, M. catarrhalis, C. diptheriae,B. pertussis, C. pneumoniae, M. pneumoniae, L. pneumophila, M.tuberculosis, B. anthracis, and Y. pestis.

In embodiments of the immunogenic combination to prevent or reduce (orto treat) infection or disease caused by a viral respiratory pathogen,the immunogenic compositions incorporate antigens selected from a virus,such as an antigen selected from: an Orthomyxovirus, such as influenzavirus, a Paramyxovirus, such Respiratory Syncytial Virus (RSV), ametapneumovirus, a parainfluenza virus (PIV), measles virus(Morbillivirus), mumps virus (Rubulavirus), Newcastle disease virus, anAdenovirus, a coronaviruses (such as SARS virus), a Rubivirus and aTogavirus.

Thus, in certain embodiments, the respiratory pathogen can be aninfluenza virus, and the protein (or peptide) antigen and the antigenencoded by the nucleic acid are antigens of influenza virus. Suitableinfluenza virus antigens include: hemagglutinin (HA), neuraminidase(NA), nucleoprotein (NP) and matrix (M) proteins. In a specificembodiment, the antigens are selected to include homologous antigens.That is, both the polypeptide antigen and the antigen encoded by thenucleic acid are both selected to be an HA antigen, an NA antigen, an NPantigen and/or an M antigen. In such a case, the HA, NA, NP and/or Mantigens can be identical, or non-identical. If non-identical, the twoantigens can nonetheless include one or more than one homologous epitopethat is identical in sequence. Alternatively, if non-identical, the twoantigens can include one or more than one homologous epitope selectedfrom different serotypes of influenza.

In certain preferred embodiments illustrated in the examples, therespiratory pathogen is a virus other than influenza, such as aParamyxovirus (e.g., RSV, hMPV or PIV). Accordingly, such an immunogeniccombination includes antigens of a Paramyxovirus. In one particularembodiment, the combination is selected to include antigens of RSV.

As indicated above, Respiratory syncytial virus (RSV) is a pathogenicvirus of the family Paramyxoviridae. Suitable antigens of RSV in thecontext of the immunogenic combinations disclosed herein can be selectedfrom: the fusion protein (F), the attachment protein (G), the matrixprotein (M2) and the nucleoprotein (N).

The term “F protein” or “Fusion protein” or “F protein polypeptide” or“Fusion protein polypeptide” refers to a polypeptide or protein havingall or part of an amino acid sequence of an RSV Fusion proteinpolypeptide. Similarly, the term “G protein” or “G protein polypeptide”refers to a polypeptide or protein having all or part of an amino acidsequence of an RSV Attachment protein polypeptide. The term “M protein”or “matrix protein” or “M protein polypeptide” refers to a polypeptideor protein having all or part of an amino acid sequence of an RSV Matrixprotein. Likewise, the term “N protein” or “Nucleocapsid protein” or “Nprotein polypeptide” refers to a polypeptide or protein having all orpart of an amino acid sequence of an RSV Nucleoprotein.

Two groups of human RSV strains have been described, the A and B groups,based mainly on differences in the antigenicity of the G glycoprotein.Numerous strains of RSV have been isolated to date, any of which aresuitable in the context of the antigens of the immunogenic combinationsdisclosed herein. Exemplary strains indicated by GenBank and/or EMBLAccession number can be found in US published application number2010/0203071 (WO2008114149), which is incorporated herein by referencefor the purpose of disclosing the nucleic acid and polypeptide sequencesof RSV F and G proteins suitable for use in the disclosed immunogeniccombinations.

Exemplary M and N protein nucleic acids and protein sequences can befound, e.g., in US published application number 2014/0141042(WO2012/089833), which are incorporated herein for purpose of disclosingthe nucleic acid and polypeptide sequences of RSV M and N proteinssuitable for use in the disclosed immunogenic combinations.

Additional strains (and their F, G, M and N protein antigens) of RSV arelikely to be isolated, and are encompassed within the genus of RSVantigens. Similarly, the genus of RSV encompasses variants arising fromnaturally occurring (e.g., previously or subsequently identifiedstrains) by genetic drift, or artificial synthesis and/or recombination.Sequences of documented RSV strain genomes and their substituent nucleicacids and the proteins encoded thereby (particularly F, G, M and Nproteins) can readily be determined by those of ordinary skill in theart by searching GenBank (on the world wide web (http://www) atncbi.nlm.nih.gov/genbank).

In certain favorable embodiments, the polypeptide antigen is an Fprotein polypeptide antigen. Particularly suitable as a polypeptideantigen component in the context of the immunogenic combinationsdisclosed herein are conformationally constrained F polypeptideantigens. Conformationally constrained F proteins have previously beendescribed in both the prefusion (PreF) and postfusion (PostF)conformations. Such conformationally constrained F proteins typicallycomprise an engineered RSV F protein ectodomain. An F protein ectodomainpolypeptide is a portion of the RSV F protein that includes all or aportion of the extracellular domain of the RSV F protein and lacks afunctional (e.g., by deletion or substitution) transmembrane domain,which can be expressed, e.g., in soluble (not attached to a membrane)form in cell culture.

Exemplary F protein antigens conformationally constrained in theprefusion conformation have been described in the art and are disclosedin detail in e.g., U.S. Pat. No. 8,563,002 (WO2009079796); US Publishedpatent application No. US2012/0093847 (WO2010/149745); US2011/0305727(WO2011/008974); US2014/0141037 and WO2012158613, each of which isincorporated herein by reference for the purpose of illustratingprefusion F polypeptides (and nucleic acids), and methods of theirproduction. Typically, the antigen is in the form of a trimer ofpolypeptides. Additional publications providing examples of F proteinsin the prefusion conformation include: McLellan et al., Science, Vol.340: 1113-1117; McLellan et al., Science, Vol 342: 592-598, and Rigteret al., PLOS One, Vol. 8: e71072, each of which can also be used in thecontext of the immunogenic combinations disclosed herein. Likewise, Fprotein antigens conformationally constrained in the postfusionconformation are also well known in the art and can be used in thecontext of the immunogenic combinations disclosed herein. Typically, theantigen is in the form of a trimer of polypeptides. Examples ofpostfusion conformationally constrained F protein polypeptides aredisclosed in detain in, e.g., US2011/0305727 (WO2011/008974), andSwanson et al., PNAS, Vol. 108:9619-9624, each of which is incorporatedherein by reference for the purpose of illustrating postfusion Fpolypeptides and nucleic acids and methods of their production.

For example, an F protein polypeptide stabilized in the prefusionconformation typically includes an ectodomain of an F protein (e.g., asoluble F protein polypeptide) comprising at least one modification thatstabilized the prefusion conformation of the F protein. For example, themodification can be selected from an addition of a trimerization domain(typically to the C terminal end), deletion of one or more of the furincleavage sites (at amino acids ˜105-109 and ˜133-136), a deletion of thepep27 domain, substitution or addition of a hydrophilic amino acid in ahydrophobic domain (e.g., HRA and/or HRB). In an embodiment, theconformationally constrained PreF antigen comprises an F2 domain (e.g.,amino acids 1-105) and an F1 domain (e.g., amino acids 137-516) of anRSV F protein polypeptide with no intervening furin cleavage sitewherein the polypeptide further comprises a heterologous trimerizationdomain positioned C-terminal to the F1 domain. Optionally, the PreFantigen also comprises a modification that alters glycosylation (e.g.,increases glycosylation), such as a substitution of one or more aminoacids at positions corresponding to amino acids ˜500-502 of an RSV Fprotein. Additionally or alternatively, the F polypeptideconformationally constrained in the prefusion conformation can includeat least two introduced cysteine residues, which are in close proximityto one another and form a disulfide bond that stabilizes the pre-fusionRSV F polypeptide. For example, the two cysteines can be within about 10Å of each other. For example, cysteines can be introduced at positions165 and 296. An exemplary PreF antigen is represented by SEQ ID NO:2.

In other embodiments, conformationally constrained F antigens caninclude one or more modifications selected from: 1) one or moremodifications (e.g., mutations) to one or both furin-cleavage sites, 2)one or more modifications to the fusion peptide, 3) one or moremodifications to the p27 linker, 4) an added oligomerization sequence;and/or an added sequence that provides a protease cleavage site.

In an embodiment, the F antigen comprises three RSV F ectodomainpolypeptides each comprising an endogenous HRA region and optionally anendogenous HRB region, and at least one oligomerization polypeptide,wherein the three ectodomain polypeptides and the at least oneoligomerization polypeptide form a six-helix bundle, with the provisothat the endogenous HRA, regions of the RSV F polypeptides are not partof the six-helix bundle. The trimer can be characterized in that thesix-helix bundle is formed with the inclusion of the HRB regions.

In one specific favorable embodiment, described in detail in theExamples, the F protein polypeptide is a protein with an amino acidsequence selected from the group of: a) a polypeptide comprising SEQ IDNO:2; b) a polypeptide with at least 80% sequence identity to SEQ IDNO:2, which polypeptide comprises an amino acid sequence correspondingto the RSV F protein polypeptide of a naturally occurring RSV strain;and c) a polypeptide with at least 95% sequence identity to SEQ ID NO:2,which polypeptide comprises an amino acid sequence that does notcorrespond to a naturally occurring RSV strain.

Methods of determining sequence identity are well known in the art, andare applicable to the foregoing antigen polypeptides, as well as thenucleic acids that encode them (e.g., as described below). Variousprograms and alignment algorithms are described in: Smith and Waterman,Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol.48:443, 1970; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp,CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881,1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988.Altschul et al., Nature Genet. 6:119, 1994, presents a detailedconsideration of sequence alignment methods and homology calculations.The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, MD)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

In some instances, the selected antigen has one or more amino acidmodification relative to the amino acid sequence of the naturallyoccurring strain from which it is derived (e.g., such as themodifications that stabilize a prefusion or postfusion conformation).Such differences can be an addition, deletion or substitution of one ormore amino acids. A variant typically differs by no more than about 1%,or 2%, or 5%, or 10%, or 15%, or 20% of the amino acid residues. Forexample, a variant antigen polypeptide sequence can include 1, or 2, orup to 5, or up to about 10, or up to about 15, or up to about 50, or upto about 100 amino acid differences as compared to the referencepolypeptide, such as the reference F antigen polypeptide sequences ofSEQ ID NO:2. Thus, a variant in the context of an RSV F protein antigentypically shares at least 80%, or 85%, more commonly, at least about 90%or more, such as 95%, or even 98% or 99% sequence identity with areference protein, e.g., the reference sequences illustrated in SEQ IDNO:2, and includes any of the exemplary PreF and/or PostF antigensdisclosed herein (e.g., by reference to U.S. Pat. No. 8,563,002(WO2009079796); US Published patent application No. US2012/0093847(WO2010/149745); US2011/0305727 (WO2011/008974); US2014/0141037;WO2012158613; McLellan et al., Science, Vol. 340: 1113-1117; McLellan etal., Science, Vol 342: 592-598; Rigter et al., PLOS One, Vol. 8: e71072;US2011/0305727 (WO2011/008974), and Swanson et al., PNAS, Vol.108:9619-9624.

Additional variants included as a feature of this disclosure are Fantigens (including PreF and PostF antigens) that include all or part ofa nucleotide or amino acid sequence selected from the naturallyoccurring variants disclosed in US published application number2010/0203071 (WO2008114149). Additional variants can arise throughgenetic drift, or can be produced artificially using site directed orrandom mutagenesis, or by recombination of two or more preexistingvariants. Such additional variants are also suitable in the context ofthe F antigens disclosed herein. For example, the modification can be asubstitution of one or more amino acids (such as two amino acids, threeamino acids, four amino acids, five amino acids, up to about ten aminoacids, or more) that do not alter the conformation or immunogenicepitopes of the resulting F (e.g., PreF or PostF) antigen.

Alternatively or additionally, the modification can include a deletionof one or more amino acids and/or an addition of one or more aminoacids. Indeed, if desired, one or more of the polypeptide domains can bea synthetic polypeptide that does not correspond to any single strain,but includes component subsequences from multiple strains, or even froma consensus sequence deduced by aligning multiple strains of RSV viruspolypeptides. For examples of consensus RSV F (as well as M and N)protein antigens, see, US 2014/0141042 (WO 2012/089833), which isincorporated herein by reference for the teaching of the design ofexemplary F, M and N consensus sequence polypeptide antigens.

In certain embodiments, one or more of the polypeptide domains ismodified by the addition of an amino acid sequence that constitutes atag, which facilitates subsequent processing or purification. Such a tagcan be an antigenic or epitope tag, an enzymatic tag or a polyhistidinetag. Typically the tag is situated at one or the other end of theprotein, such as at the C-terminus or N-terminus of the antigen orfusion protein.

In addition to a polypeptide or peptide antigen, the immunogeniccombinations disclosed herein also include a second immunogeniccomponent that contains a nucleic acid that encodes an antigen of thesame respiratory pathogen (as the peptide or polypeptide antigencontained in the first immunogenic component). It is contemplated thatthe nucleic acid is a nucleic acid other than a plasmid DNA. The nucleicacid can be in the form of a replicating or replication defectivevector, such as a viral vector. Numerous viral vectors suitable forintroducing immunogenic nucleic acids into a subject are known in theart, and include both DNA and RNA viruses. Suitable examples forencoding an antigen in the context of the immunogenic combinationsdisclosed herein include, for example: adenovirus vectors (replicatingor replication deficient), pox virus vectors, including vaccinia virusvectors, such as modified vaccinia Ankara virus (MVA), NYVAC, avipoxvectors, canarypox (ALVAC) and fowl pox virus (FPV), Alphavirus vectors(such as Sindbis virus, Semlike Forest virus (SFV), Ross River virus,and Venezuelan equine encephalitis (VEE) virus) and chimeras andreplicons thereof, herpes virus vectors (e.g., cytomegalovirus(CMV)-derived vectors), arena virus vectors, such as lymphocyticchoriomeningitis virus (LCMV) vectors, measles virus vectors, vesicularstomatitis virus vectors, pseudorabies virus, adeno-associated virus,retrovirus, lentivirus, viral like particles, and many others.

In one particular embodiment, the vector is an adenovirus. Theproduction and use of Adenovirus vectors are well known to those ofordinary skill in the art. In the context of the immunogeniccombinations disclosed here, examples of disclosure of the design,production and use of adenovirus vectors containing antigens of arespiratory pathogen can be found in, e.g., US published application no.US2014/0141042 (WO 2012/089833). Additional detail concerning theadenovirus vectors is found, e.g., in U.S. Pat. No. 8,216,834 (WO2005/071093); and US published application no. US2012/0027788 (WO2010/086189); US published application no. US20050214323.

Adenoviral vectors of use in the present invention may be derived from arange of mammalian hosts.

Over 100 distinct serotypes of adenovirus have been isolated whichinfect various mammalian species, 51 of which are of human origin. Thusone or more of the adenoviral vectors may be derived from a humanadenovirus. Examples of such human-derived adenoviruses are Ad1, Ad2,Ad4, Ad5, Ad6, Ad11, Ad 24, Ad34, Ad35, particularly Ad5, Ad11 and Ad35.The human and nonhuman adenviral serotypes have been categorised intosix subgenera (A-F) based on a number of biological, chemical,immunological and structural criteria.

Although Ad5-based vectors have been used extensively in a number ofgene therapy trials, there may be limitations on the use of Ad5 andother human group C adenoviral vectors due to preexisting immunity inthe general population due to natural infection. Ad5 and other humangroup C members tend to be among the most seroprevalent serotypes.Immunity to existing vectors may develop as a result of exposure to thevector during treatment. These types of preexisting or developedimmunity to seroprevalent vectors may limit the effectiveness of genetherapy or vaccination efforts. Alternative adenovirus serotypes, thusconstitute very important targets in the pursuit of gene deliverysystems capable of evading the host immune response.

One such area of alternative serotypes are those derived from non humanprimates, especially adenoviruses isolated from chimpanzee, bonobos andgorillas. See U.S. Pat. No. 6,083,716 which describes the genome of twochimpanzee adenoviruses.

It has been shown that nonhuman simian adenoviral vectors induce strongimmune responses to transgene products as efficiently as humanadenoviral vectors (Fitzgerald et al. J. Immunol. 170:1416; (Colloca etal. (2012) Science Translational Medicine 4:1-9; Roy et al. (2004)Virology 324: 361-372; Roy et al. (2010) Journal of Gene Medicine13:17-25).

Non human primate adenoviruses can be isolated from the mesenteric lymphnodes or feces of the animals and can replicate in vitro in HEK 293cells. Despite these similarities, nonhuman simian adenoviruses arephylogenetically and immunologically distinct from the more common humanserotypes (Ad2 and Ad5).

Thus one or more of the adenoviral vectors may be derived from anon-human primate adenovirus eg a chimpanzee adenovirus such as oneselected from serotypes ChAd3, ChAd63, ChAd83, ChAd155, Pan5, Pan6, Pan7and Pan9. Specifically, the virus may be a non-human adenovirus, such asa simian adenovirus and in particular a chimpanzee adenovirus such asChAd155, Pan 5, 6, 7 or 9. Examples of such strains are described inWO03/000283 and are available from the American Type Culture Collection,10801 University Boulevard, Manassas, Virginia 20110-2209, and othersources. Desirable chimpanzee adenovirus strains include Pan 5 [ATCCVR-591], Pan 6 [ATCC VR-592], and Pan 7 [ATCC VR-593]. Alternatively,adenoviral vectors may be derived from nonhuman simian adenovirusesderived from bonobos, such as PanAd1, PanAd2 or PanAd3. Examples of suchvectors described herein can be found for example in WO2005/071093,WO2010/086189 and GB1510357.5.

Use of nonhuman simian adenoviruses is thought to be advantageous overuse of human adenovirus serotypes because of the lack of pre-existingimmunity, in particular the lack of cross-neutralising antibodies, toadenoviruses in the target population. Cross-reaction of the chimpanzeeadenoviruses with pre-existing neutralizing antibody responses is onlypresent in 2% of the target population compared with 35% in the case ofcertain candidate human adenovirus vectors. The chimpanzee adenovirusesare distinct from the more common human subtypes Ad2 and Ad5, but aremore closely related to human Ad4 of subgroup E, which is not aprevalent subtype. Pan 6 is less closely related to Pan 5, 7 and 9.

The adenovirus of the invention may be replication defective. This meansthat it has a reduced ability to replicate in non-complementing cells,compared to the wild type virus. This may be brought about by mutatingthe virus e.g. by deleting a gene involved in replication, for exampledeletion of the E1a, E1b, E3 or E4 gene.

The adenoviral vectors in accordance with the present invention may bederived from replication defective adenovirus comprising a functional E1deletion. Thus the adenoviral vectors according to the invention may bereplication defective due to the absence of the ability to expressadenoviral E1a and E1b, i.e., are functionally deleted in E1a and E1b.The recombinant adenoviruses may also bear functional deletions in othergenes [see WO 03/000283] for example, deletions in E3 or E4 genes. Theadenovirus delayed early gene E3 may be eliminated from the adenovirussequence which forms part of the recombinant virus. The function of E3is not necessary to the production of the recombinant adenovirusparticle. Thus, it is unnecessary to replace the function of this geneproduct in order to package a recombinant adenovirus useful in theinvention. In one particular embodiment the recombinant adenoviruseshave functionally deleted E1 and E3 genes. The construction of suchvectors is described in Roy et al., Human Gene Therapy 15:519-530, 2004.

Recombinant adenoviruses may also be constructed having a functionaldeletion of the E4 gene, although it may be desirable to retain the E4ORF6 function. Adenovirus vectors according to the invention may alsocontain a deletion in the delayed early gene E2a. Deletions may also bemade in any of the late genes L1 through to L5 of the adenovirus genome.Similarly deletions in the intermediate genes IX and IVa may be useful.

Other deletions may be made in the other structural or non-structuraladenovirus genes. The above deletions may be used individually, i.e. anadenovirus sequence for use in the present invention may containdeletions of E1 only. Alternatively, deletions of entire genes orportions thereof effective to destroy their biological activity may beused in any combination. For example in one exemplary vector, theadenovirus sequences may have deletions of the E1 genes and the E4 gene,or of the E1, E2a and E3 genes, or of the E1 and E3 genes (such asfunctional deletions in E1a and E1b, and a deletion of at least part ofE3), or of the E1, E2a and E4 genes, with or without deletion of E3 andso on. Such deletions may be partial or full deletions of these genesand may be used in combination with other mutations, such as temperaturesensitive mutations to achieve a desired result. Adenoviral vectors ofuse in the present invention include PanAd3 (WO 2010/086189) and ChAd155(GB1510357.5).

The adenoviral vectors can be produced on any suitable cell line inwhich the virus is capable of replication. In particular, complementingcell lines which provide the factors missing from the viral vector thatresult in its impaired replication characteristics (such as E1 and/orE4) can be used. Without limitation, such a cell line may be HeLa [ATCCAccession No. CCL 2], A549 [ATCC Accession No. CCL 185], HEK 293, KB[CCL 17], Detroit [e.g., Detroit 510, CCL 72] and WI-38 [CCL 75] cells,among others. These cell lines are all available from the American TypeCulture Collection, 10801 University Boulevard, Manassas, Virginia20110-2209. Other suitable parent cell lines may be obtained from othersources, such as PER.C6© cells, as represented by the cells depositedunder ECACC no. 96022940 at the European Collection of Animal CellCultures (ECACC) at the Centre for Applied Microbiology and Research(CAMR, UK) or Her 96 cells (Crucell).

In another embodiment, the viral vector is a pox virus vector. Infavorable embodiments the pox virus is selected from the group of: U.S.Pat. No. 6,761,893 (WO02/42480); U.S. Pat. Nos. 7,964,395; 7,964,396; USpublished application no. US2013/0183335 (WO2012/048817); and PCTpublished application no. WO2014/019718 provide exemplary vectors andmethods for the production of MVA vectors suitable in the context of animmunogenic component as disclosed herein. Each of the preceding isincorporated herein by reference for the teaching of suitable MVAvectors and methods.

In another embodiment, the viral vector is an Alphavirus vector, such asan alphavirus replicon or other self-replicating RNA vector. Exemplaryalphavirus vectors and methods for producing and delivering themsuitable for use in the context of the immunogenic combinationsdisclosed herein are described in, e.g., US20090104226 (WO2006078294);US20110300205 (WO2011005799); US20130195968 (WO 2012/006376);US20130177639 (WO2012006377); WO2013006838; and WO2013006842, each ofwhich are incorporated herein for their disclosure of exemplaryself-replicating RNA vectors suitable in the context of the disclosedimmunogenic combinations.

In the context of the immunogenic combinations disclosed herein, thepolypeptide antigen of the respiratory pathogen, and the antigen of thesame pathogen encoded by a nucleic acid can be the same or different.Favorably, in the context of the immunogenic combinations disclosedherein the two immunogenic components are homologous (that is related bydescent from a common evolutionary precursor), and thus share at leastpartial sequence identity (as determined above). In some embodiments,the first immunogenic component and the antigen(s) encoded by the secondimmunogenic component of the immunogenic combination include at leastone identical or homologous antigen. In certain embodiments, theantigens are non-identical, in which case, the antigens can comprisepartially identical amino acid sequences, for example, such that theyinclude at least one identical or partially identical immunogenicepitope. Partially identical epitopes can be, for example, selected froma corresponding portion of a homologous antigen of another strain orserotype of the pathogen.

In certain embodiments of the immunogenic combination, the antigen ofthe first component and the antigen encoded by the nucleic acid of thesecond component share substantial sequence identity, such as about 70%sequence identity across all or a portion of their length, for example,about 75% identity, about 80% identity, about 85% identity, about 90%identity or about or greater than 95% identity. In an embodiment inwhich one component includes (or encodes) one antigen, and the othercomponent includes (or encodes) multiple antigens, the sequence identityis compared between the corresponding antigens. To illustrate, in anembodiment in which the first component contains an RSV F proteinantigen, and the second component contains a nucleic acid that encodesan RSV F antigen and RSV, M and N antigens, the sequence identity iscompared between the F proteins (without regard to the M and N proteincomponents). Where multiple antigens are included in each component, atleast one is expected to meet the threshold of 70% sequence identity.

For example, in one favorable embodiment to elicit an immune responsespecific for RSV, the immunogenic combination includes a firstimmunogenic component that contains an RSV F protein antigen. The secondimmunogenic component contains a nucleic acid that encodes an RSV Fprotein. In an embodiment, one or both of the immunogenic components canbe an ectodomain of an RSV F Protein (FΔTM). In an embodiment, theantigens are identical. In another embodiment, the F protein antigensare non-identical in sequence. For example, in one exemplary embodimentdescribed in more detail below, the first immunogenic component includesa polypeptide antigen that is a conformationally constrained F proteinantigen, and the second immunogenic component includes a nucleic acid(for example, and adenovirus vector) that encodes an F proteinpolypeptide of a different sequence that is not conformationallyconstrained, e.g., a consensus sequence F protein polypeptide designedas described in US2012/0027788. In one embodiment, the first componentcontains an RSV F protein antigen, and the second component contains anucleic acid that encodes an RSV F antigen and RSV, M and N antigens.More specifically, the first component contains an RSV F protein antigenconformationally constrained in the prefusion conformation, and thesecond component contains a nucleic acid that encodes an RSV FΔTMantigen and RSV M2-1 and N antigens, wherein a self-cleavage site isincluded between the RSV FΔTM antigen and the RSV M2-1 and a flexiblelinker is included between the RSV M2-1 and N antigens. Morespecifically, the first component may contain an RSV F protein antigenrepresented by SEQ ID NO:2, and the second component may contain anadenoviral vector carrying a nucleic acid insert represented by SEQ IDNO:3.

Optionally, one or both of the immunogenic components includes aplurality of antigens. Typically, these are selected from the sametarget pathogen. However, embodiments are contemplated in which antigensof multiple pathogens are included in the immunogenic combination.

Immunogenic Components and Combinations

In the context of the immunogenic combinations disclosed herein, theimmunogenic components comprising (nucleic acid and protein) can beformulated for administration in a single immunogenic composition or indifferent immunogenic compositions. When formulated for administrationin a single composition the components can be admixed prior toadministration or stably co-formulated during manufacture.

The immunogenic compositions disclosed herein typically contain apharmaceutically acceptable carrier or excipients. Pharmaceuticallyacceptable carriers and excipients are well known and can be selected bythose of skill in the art. The adjective “pharmaceutically acceptable”indicates that the referent is suitable for administration to a subject(e.g., a human or animal subject). Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975),describes compositions and formulations (including diluents) suitablefor pharmaceutical delivery of therapeutic and/or prophylacticcompositions, including immunogenic compositions.

For example, in the context of the immunogenic components andcombinations disclosed herein, the carrier or excipient can favorablyinclude a buffer. Optionally, the carrier or excipient also contains atleast one component that stabilizes solubility and/or stability.Examples of solubilizing/stabilizing agents include detergents, forexample, laurel sarcosine and/or tween. Alternativesolubilizing/stabilizing agents include arginine, and glass formingpolyols (such as sucrose, trehalose and the like). Numerouspharmaceutically acceptable carriers and/or pharmaceutically acceptableexcipients are known in the art and are described, e.g., in Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,PA, 5th Edition (975).

Accordingly, suitable excipients and carriers can be selected by thoseof skill in the art to produce a formulation suitable for delivery to asubject by a selected route of administration.

Suitable excipients include, without limitation: glycerol, Polyethyleneglycol (PEG), Sorbitol, Trehalose, N-lauroylsarcosine sodium salt,L-proline, Non detergent sulfobetaine, Guanidine hydrochloride, Urea,Trimethylamine oxide, KCl, Ca2+, Mg2+, Mn2+, Zn2+ and other divalentcation related salts, Dithiothreitol, Dithioerytrol, andβ-mercaptoethanol. Other excipients can be detergents (including:Tween80, Tween20, Triton X-00, NP-40, Empigen BB, Octylglucoside,Lauroyl maltoside, Zwittergent 3-08, Zwittergent 3-0, Zwittergent 3-2,Zwittergent 3-4, Zwittergent 3-6, CHAPS, Sodium deoxycholate, Sodiumdodecyl sulphate, Cetyltrimethylammonium bromide).

Optionally, the disclosed immunogenic combinations also include anadjuvant, which adjuvant also may be used with the disclosed vaccineregimens, methods, uses and kits. In certain embodiments, theimmunogenic component containing the polypeptide antigen of arespiratory pathogen is formulated with an adjuvant. In otherembodiments, the immunogenic component containing the nucleic acid thatencodes a respiratory pathogen antigen is formulated with an adjuvant.In an embodiment, both immunogenic components are administered in acomposition containing an adjuvant. Typically, the adjuvant is admixed(e.g., prior to administration or stably formulated) with the antigeniccomponent. When the combination immunogenic composition is to beadministered to a subject of a particular age group, the adjuvant isselected to be safe and effective in the subject or population ofsubjects. Thus, when formulating a combination immunogenic compositionfor administration in an elderly subject (such as a subject greater than65 years of age), the adjuvant is selected to be safe and effective inelderly subjects. Similarly, when the combination immunogeniccomposition is intended for administration in neonatal or infantsubjects (such as subjects between birth and the age of two years), theadjuvant is selected to be safe and effective in neonates and infants.In the case of an adjuvant selected for safety and efficacy in neonatesand infants, an adjuvant dose can be selected that is a dilution (e.g.,a fractional dose) of a dose typically administered to an adult subject.

Additionally, the adjuvant is typically selected to enhance the desiredaspect of the immune response when administered via a route ofadministration, by which the combination immunogenic composition isadministered. For example, when formulating a combination immunogeniccomposition for nasal administration, proteosome and protollin arefavorable adjuvants. In contrast, when the combination immunogeniccomposition is formulated for intramuscular administration, adjuvantsincluding one or more of 3D-MPL, squalene (e.g., QS21), liposomes,and/or oil and water emulsions are favorably selected.

One suitable adjuvant for use with the immunogenic combinationsdisclosed herein is a non-toxic bacterial lipopolysaccharide derivative.An example of a suitable non-toxic derivative of lipid A, ismonophosphoryl lipid A or more particularly 3-Deacylated monophosphoryllipid A (3D-MPL). 3D-MPL is sold under the name MPL by GlaxoSmithKlineBiologicals N.A., and is referred throughout the document as MPL or3D-MPL. See, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034and 4,912,094. 3D-MPL primarily promotes CD4+ T cell responses with anIFN-γ (Th1) phenotype. 3D-MPL can be produced according to the methodsdisclosed in GB2220211 A. Chemically it is a mixture of 3-deacylatedmonophosphoryl lipid A with 3, 4, 5 or 6 acylated chains. In thecompositions of the present disclosure small particle 3D-MPL can beused. Small particle 3D-MPL has a particle size such that it can besterile-filtered through a 0.22 μm filter. Such preparations aredescribed in WO94/21292.

A lipopolysaccharide, such as 3D-MPL, can be used at amounts between 1and 50 μg, per human dose of the immunogenic composition. Such 3D-MPLcan be used at a level of about 25 μg, for example between 20-30 μg,suitably between 21-29 μg or between 22 and 28 μg or between 23 and 27μg or between 24 and 26 μg, or 25 μg. In another embodiment, the humandose of the immunogenic composition comprises 3D-MPL at a level of about10 μg, for example between 5 and 15 μg, suitably between 6 and 14 μg,for example between 7 and 13 μg or between 8 and 12 μg or between 9 and11 μg, or 10 μg. In a further embodiment, the human dose of theimmunogenic composition comprises 3D-MPL at a level of about 5 μg, forexample between 1 and 9 μg, or between 2 and 8 μg or suitably between 3and 7 μg or 4 and μg, or 5 μg.

In other embodiments, the lipopolysaccharide can be a β(1-6) glucosaminedisaccharide, as described in U.S. Pat. No. 6,005,099 and EP Patent No.0 729 473 B1. One of skill in the art would be readily able to producevarious lipopolysaccharides, such as 3D-MPL, based on the teachings ofthese references. Nonetheless, each of these references is incorporatedherein by reference. In addition to the aforementioned immunostimulants(that are similar in structure to that of LPS or MPL or 3D-MPL),acylated monosaccharide and disaccharide derivatives that are asub-portion to the above structure of MPL are also suitable adjuvants.In other embodiments, the adjuvant is a synthetic derivative of lipid A,some of which are described as TLR-4 agonists, and include, but are notlimited to: OM174(2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-o-phosphono-β-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranosyldihydrogenphosphate),(WO 95/14026); OM 294 DP (3S, 9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)-3-hydroxytetradecanoylamino]decan-1,10-diol,1,10-bis(dihydrogenophosphate)(WO 99/64301 and WO 00/0462); and OM 197 MP-Ac DP (3S—,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]decan-1,10-diol,1-dihydrogenophosphate10-(6-aminohexanoate) (WO 01/46127).

Other TLR4 ligands which can be used are alkyl Glucosaminide phosphates(AGPs) such as those disclosed in WO 98/50399 or U.S. Pat. No. 6,303,347(processes for preparation of AGPs are also disclosed), suitably RC527or RC529 or pharmaceutically acceptable salts of AGPs as disclosed inU.S. Pat. No. 6,764,840. Some AGPs are TLR4 agonists, and some are TLR4antagonists. Both are thought to be useful as adjuvants.

Other suitable TLR-4 ligands, capable of causing a signaling responsethrough TLR-4 (Sabroe et al, JI 2003 p 1630-5) are, for example,lipopolysaccharide from gram-negative bacteria and its derivatives, orfragments thereof, in particular a non-toxic derivative of LPS (such as3D-MPL). Other suitable TLR agonists are: heat shock protein (HSP) 10,60, 65, 70, 75 or 90; surfactant Protein A, hyaluronan oligosaccharides,heparan sulphate fragments, fibronectin fragments, fibrinogen peptidesand b-defensin-2, and muramyl dipeptide (MDP). In one embodiment the TLRagonist is HSP 60, 70 or 90. Other suitable TLR-4 ligands are asdescribed in WO 2003/011223 and in WO 2003/099195, such as compound I,compound II and compound III disclosed on pages 4-5 of WO2003/011223 oron pages 3-4 of WO2003/099195 and in particular those compoundsdisclosed in WO2003/011223 as ER803022, ER803058, ER803732, ER804053,ER804057, ER804058, ER804059, ER804442, ER804680, and ER804764. Forexample, one suitable TLR-4 ligand is ER804057.

Additional TLR agonists are also useful as adjuvants. The term “TLRagonist” refers to an agent that is capable of causing a signalingresponse through a TLR signaling pathway, either as a direct ligand orindirectly through generation of endogenous or exogenous ligand. Suchnatural or synthetic TLR agonists can be used as alternative oradditional adjuvants. A brief review of the role of TLRs as adjuvantreceptors is provided in Kaisho & Akira, Biochimica et Biophysica Acta1589:1-13, 2002. These potential adjuvants include, but are not limitedto agonists for TLR2, TLR3, TLR7, TLR8 and TLR9. Accordingly, in oneembodiment, the adjuvant and combination immunogenic composition furthercomprises an adjuvant which is selected from the group consisting of: aTLR-1 agonist, a TLR-2 agonist, TLR-3 agonist, a TLR-4 agonist, TLR-5agonist, a TLR-6 agonist, TLR-7 agonist, a TLR-8 agonist, TLR-9 agonist,or a combination thereof.

In one embodiment of the present disclosure, a TLR agonist is used thatis capable of causing a signaling response through TLR-1. Suitably, theTLR agonist capable of causing a signaling response through TLR-1 isselected from: Tri-acylated lipopeptides (LPs); phenol-soluble modulin;Mycobacterium tuberculosis LP;S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys(4)-OH,trihydrochloride (Pam3Cys) LP which mimics the acetylated amino terminusof a bacterial lipoprotein and OspA LP from Borrelia burgdorferi.

In an alternative embodiment, a TLR agonist is used that is capable ofcausing a signaling response through TLR-2. Suitably, the TLR agonistcapable of causing a signaling response through TLR-2 is one or more ofa lipoprotein, a peptidoglycan, a bacterial lipopeptide from Mtuberculosis, B burgdorferi or T pallidum; peptidoglycans from speciesincluding Staphylococcus aureus; lipoteichoic acids, mannuronic acids,Neisseria porins, bacterial fimbriae, Yersina virulence factors, CMVvirions, measles haemagluttinin, and zymosan from yeast.

In an alternative embodiment, a TLR agonist is used that is capable ofcausing a signaling response through TLR-3. Suitably, the TLR agonistcapable of causing a signaling response through TLR-3 is double strandedRNA (dsRNA), or polyinosinic-polycytidylic acid (Poly IC), a molecularnucleic acid pattern associated with viral infection.

In an alternative embodiment, a TLR agonist is used that is capable ofcausing a signaling response through TLR-5. Suitably, the TLR agonistcapable of causing a signaling response through TLR-5 is bacterialflagellin.

In an alternative embodiment, a TLR agonist is used that is capable ofcausing a signaling response through TLR-6. Suitably, the TLR agonistcapable of causing a signaling response through TLR-6 is mycobacteriallipoprotein, di-acylated LP, and phenol-soluble modulin. Additional TLR6agonists are described in WO 2003/043572.

In an alternative embodiment, a TLR agonist is used that is capable ofcausing a signaling response through TLR-7. Suitably, the TLR agonistcapable of causing a signaling response through TLR-7 is a singlestranded RNA (ssRNA), loxoribine, a guanosine analogue at positions N7and C8, or an imidazoquinoline compound, or derivative thereof. In oneembodiment, the TLR agonist is imiquimod. Further TLR7 agonists aredescribed in WO 2002/085905.

In an alternative embodiment, a TLR agonist is used that is capable ofcausing a signaling response through TLR-8. Suitably, the TLR agonistcapable of causing a signaling response through TLR-8 is a singlestranded RNA (ssRNA), an imidazoquinoline molecule with anti-viralactivity, for example resiquimod (R848); resiquimod is also capable ofrecognition by TLR-7. Other TLR-8 agonists which can be used includethose described in WO 2004/071459.

In an alternative embodiment, a TLR agonist is used that is capable ofcausing a signaling response through TLR-9. In one embodiment, the TLRagonist capable of causing a signaling response through TLR-9 is HSP90.Alternatively, the TLR agonist capable of causing a signaling responsethrough TLR-9 is bacterial or viral DNA, DNA containing unmethylated CpGnucleotides, in particular sequence contexts known as CpG motifs.CpG-containing oligonucleotides induce a predominantly Th1 response.Such oligonucleotides are well known and are described, for example, inWO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462.Suitably, CpG nucleotides are CpG oligonucleotides. Suitableoligonucleotides for use in the combination immunogenic composition areCpG containing oligonucleotides, optionally containing two or moredinucleotide CpG motifs separated by at least three, suitably at leastsix or more nucleotides. A CpG motif is a Cytosine nucleotide followedby a Guanine nucleotide. The CpG oligonucleotides are typicallydeoxynucleotides. In a specific embodiment the internucleotide in theoligonucleotide is phosphorodithioate, or suitably a phosphorothioatebond, although phosphodiester and other internucleotide bonds arepossible. Also possible are oligonucleotides with mixed internucleotidelinkages. Methods for producing phosphorothioate oligonucleotides orphosphorodithioate are described in U.S. Pat. Nos. 5,666,153, 5,278,302and WO 95/26204.

Other adjuvants that can be used in the disclosed immunogeniccombinations, and with the disclosed immunization regimens, methods,uses and kits, e.g., on their own or in combination with 3D-MPL, oranother adjuvant described herein, are saponins, such as QS21.

Saponins are taught in: Lacaille-Dubois, M and Wagner H. (1996. A reviewof the biological and pharmacological activities of saponins.Phytomedicine vol 2 pp 363-386). Saponins are steroid or triterpeneglycosides widely distributed in the plant and marine animal kingdoms.Saponins are noted for forming colloidal solutions in water which foamon shaking, and for precipitating cholesterol. When saponins are nearcell membranes they create pore-like structures in the membrane whichcause the membrane to burst. Haemolysis of erythrocytes is an example ofthis phenomenon, which is a property of certain, but not all, saponins.

Saponins are known as adjuvants in vaccines for systemic administration.The adjuvant and haemolytic activity of individual saponins has beenextensively studied in the art (Lacaille-Dubois and Wagner, supra). Forexample, Quil A (derived from the bark of the South American treeQuillaja Saponaria Molina), and fractions thereof, are described in U.S.Pat. No. 5,057,540 and “Saponins as vaccine adjuvants”, Kensil, C. R.,Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2):1-55; and EP 0 362 279B1. Particulate structures, termed Immune Stimulating Complexes(ISCOMS), comprising fractions of Quil A are haemolytic and have beenused in the manufacture of vaccines (Morein, B., EP 0 109 942 B1; WO96/11711; WO 96/33739). The haemolytic saponins QS21 and QS17 (HPLCpurified fractions of Quil A) have been described as potent systemicadjuvants, and the method of their production is disclosed in U.S. Pat.No. 5,057,540 and EP 0 362 279 B1, which are incorporated herein byreference. Other saponins which have been used in systemic vaccinationstudies include those derived from other plant species such asGypsophila and Saponaria (Bomford et al., Vaccine, 10(9):572-577, 1992).

QS21 is an Hplc purified non-toxic fraction derived from the bark ofQuillaja Saponaria Molina. A method for producing QS21 is disclosed inU.S. Pat. No. 5,057,540. Non-reactogenic adjuvant formulationscontaining QS21 are described in WO 96/33739. The aforementionedreferences are incorporated by reference herein. Said immunologicallyactive saponin, such as QS21, can be used in amounts of between 1 and 50μg, per human dose of the combination immunogenic composition.Advantageously QS21 is used at a level of about 25 μg, for examplebetween 20-30 μg, suitably between 21-29 μg or between 22-28 μg orbetween 23-27 μg or between 24-26 μg, or 25 μg. In another embodiment,the human dose of the combination immunogenic composition comprises QS21at a level of about 10 μg, for example between 5 and 15 μg, suitablybetween 6-14 μg, for example between 7-13 μg or between 8-12 μg orbetween 9-11 μg, or 10 μg. In a further embodiment, the human dose ofthe combination immunogenic composition comprises QS21 at a level ofabout 5 μg, for example between 1-9 μg, or between 2-8 μg or suitablybetween 3-7 μg or 4-6 μg, or 5 μg. Such formulations comprising QS21 andcholesterol have been shown to be successful adjuvants when formulatedtogether with an antigen. Thus, for example, polypeptides of thedisclosed immunogenic combinations can be provided with an adjuvantcomprising a combination of QS21 and cholesterol.

Optionally, the adjuvant can alternatively or additionally includemineral salts such as an aluminium salt (for example, aluminumhydroxide, aluminum phosphate, aluminum potassium sulfate, aluminumhydroxyphosphate sulfate) or calcium salt (for example, calciumhydroxide, calcium fluoride, calcium phosphate). Other salts suitable inthe formulation of an adjuvant include cerium(III) nitrate hexahydrateand zinc sulfate heptahydrate. For example, an adjuvant containing3D-MPL in combination with an aluminium salt (e.g., aluminium hydroxideor “alum”) is suitable for formulation in a combination immunogeniccombinations containing antigen(s) of a respiratory pathogen as descriedherein. Alternatively, such mineral salt adjuvants may be used otherthan in combination with non-mineral-salt adjuvants, i.e. thecombination immunogenic composition may be adjuvanted only with one, ormore than one, mineral salt adjuvant such as aluminium hydroxide,aluminium phosphate and calcium phosphate, etc.

Another class of suitable adjuvants for use in the immunogeniccombinations disclosed herein includes OMP-based immunostimulatorycompositions. OMP-based immunostimulatory compositions are particularlysuitable as mucosal adjuvants, e.g., for intranasal administration.OMP-based immunostimulatory compositions are a genus of preparations ofouter membrane proteins (OMPs, including some porins) from Gram-negativebacteria, such as, but not limited to, Neisseria species (see, e.g.,Lowell et al., J. Exp. Med. 167:658, 1988; Lowell et al., Science240:800, 1988; Lynch et al., Biophys. J. 45:104, 1984; Lowell, in “NewGeneration Vaccines” 2nd ed., Marcel Dekker, Inc., New York, Basil, HongKong, page 193, 1997; U.S. Pat. Nos. 5,726,292; 4,707,543), which areuseful as a carrier or in compositions for immunogens, such as bacterialor viral antigens. Some OMP-based immunostimulatory compositions can bereferred to as “Proteosomes,” which are hydrophobic and safe for humanuse. Proteosomes have the capability to auto-assemble into vesicle orvesicle-like OMP clusters of about 20 nm to about 800 nm, and tononcovalently incorporate, coordinate, associate (e.g.,electrostatically or hydrophobically), or otherwise cooperate withprotein antigens (Ags), particularly antigens that have a hydrophobicmoiety. Any preparation method that results in the outer membraneprotein component in vesicular or vesicle-like form, includingmulti-molecular membranous structures or molten globular-like OMPcompositions of one or more OMPs, is included within the definition ofProteosome. Proteosomes can be prepared, for example, as described inthe art (see, e.g., U.S. Pat. No. 5,726,292 or 5,985,284). Proteosomescan also contain an endogenous lipopolysaccharide or lipooligosaccharide(LPS or LOS, respectively) originating from the bacteria used to producethe OMP porins (e.g., Neisseria species), which generally will be lessthan 2% of the total OMP preparation.

Proteosomes are composed primarily of chemically extracted outermembrane proteins (OMPs) from Neisseria menigitidis (mostly porins A andB as well as class 4 OMP), maintained in solution by detergent (LowellGH. Proteosomes for Improved Nasal, Oral, or Injectable Vaccines. In:Levine M M, Woodrow G C, Kaper J B, Cobon G S, eds, New GenerationVaccines. New York: Marcel Dekker, Inc. 1997; 193-206). Proteosomes canbe formulated with a variety of antigens such as purified or recombinantproteins derived from viral sources, including the RSV F proteinpolypeptides disclosed herein, e.g., by diafiltration or traditionaldialysis processes or with purified B. pertussis antigenic proteins. Thegradual removal of detergent allows the formation of particulatehydrophobic complexes of approximately 100-200 nm in diameter (Lowell GH. Proteosomes for Improved Nasal, Oral, or Injectable Vaccines. In:Levine M M, Woodrow G C, Kaper J B, Cobon G S, eds, New GenerationVaccines. New York: Marcel Dekker, Inc. 1997; 193-206).

“Proteosome: LPS or Protollin” as used herein refers to preparations ofproteosomes admixed, e.g., by the exogenous addition, with at least onekind of lipo-polysaccharide to provide an OMP-LPS composition (which canfunction as an immunostimulatory composition). Thus, the OMP-LPScomposition can be comprised of two of the basic components ofProtollin, which include (1) an outer membrane protein preparation ofProteosomes (e.g., Projuvant) prepared from Gram-negative bacteria, suchas Neisseria meningitidis, and (2) a preparation of one or moreliposaccharides. A lipo-oligosaccharide can be endogenous (e.g.,naturally contained with the OMP Proteosome preparation), can be admixedor combined with an OMP preparation from an exogenously preparedlipo-oligosaccharide (e.g., prepared from a different culture ormicroorganism than the OMP preparation), or can be a combinationthereof. Such exogenously added LPS can be from the same Gram-negativebacterium from which the OMP preparation was made or from a differentGram-negative bacterium. Protollin should also be understood tooptionally include lipids, glycolipids, glycoproteins, small molecules,or the like, and combinations thereof. The Protollin can be prepared,for example, as described in U.S. Patent Application Publication No.2003/0044425.

Combinations of different adjuvants, such as those mentionedhereinabove, can also be used in the disclosed immunogenic combinations(e.g., with individual components or admixtures thereof). For example,as already noted, QS21 can be formulated together with 3D-MPL. The ratioof QS21:3D-MPL will typically be in the order of 1:10 to 10:1; such as1:5 to 5:1, and often substantially 1:1. Typically, the ratio is in therange of 2.5:1 to 1:1 3D-MPL: QS21. Another combination adjuvantformulation includes 3D-MPL and an aluminium salt, such as aluminiumhydroxide.

In some instances, the adjuvant formulation includes a mineral salt,such as an aluminium (alum) salt for example aluminium phosphate oraluminium hydroxide, or calcium phosphate. Where alum is present, e.g.,in combination with 3D-MPL, the amount is typically between about 100 μgand 1 mg, such as from about 100 μg, or about 200 μg to about 750 μg,such as about 500 μg per dose.

In some embodiments, the adjuvant includes an oil and water emulsion,e.g., an oil-in-water emulsion. One example of an oil-in-water emulsioncomprises a metabolisable oil, such as squalene, a tocol such as atocopherol, e.g., alpha-tocopherol, and a surfactant, such as sorbitantrioleate (Span 85™) or polyoxyethylene sorbitan monooleate (Tween 80™),in an aqueous carrier. In certain embodiments, the oil-in-water emulsiondoes not contain any additional immunostimulants(s), (in particular itdoes not contain a non-toxic lipid A derivative, such as 3D-MPL, or asaponin, such as QS21). The aqueous carrier can be, for example,phosphate buffered saline. Additionally the oil-in-water emulsion cancontain span 85 and/or lecithin and/or tricaprylin.

In another embodiment the combination immunogenic composition comprisesan oil-in-water emulsion and optionally one or more furtherimmunostimulants, wherein said oil-in-water emulsion comprises 0.5-10 mgmetabolisable oil (suitably squalene), 0.5-11 mg tocol (suitably atocopherol, such as alpha-tocopherol) and 0.4-4 mg emulsifying agent.

In one specific embodiment, the adjuvant formulation includes 3D-MPLprepared in the form of an emulsion, such as an oil-in-water emulsion.In some cases, the emulsion has a small particle size of less than 0.2μm in diameter, as disclosed in WO 94/21292. For example, the particlesof 3D-MPL can be small enough to be sterile filtered through a 0.22micron membrane (as described in European Patent number 0 689 454).Alternatively, the 3D-MPL can be prepared in a liposomal formulation.Optionally, the adjuvant containing 3D-MPL (or a derivative thereof)also includes an additional immunostimulatory component.

The adjuvant is selected to be safe and effective in the population towhich the immunogenic composition is administered. For adult and elderlypopulations, the formulations typically include more of an adjuvantcomponent than is typically found in an infant formulation. Inparticular formulations using an oil-in-water emulsion, such an emulsioncan include additional components, for example, such as cholesterol,squalene, alpha tocopherol, and/or a detergent, such as tween 80 orspan85. In exemplary formulations, such components can be present in thefollowing amounts: from about 1-50 mg cholesterol, from 2 to 10%squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween 80.Typically, the ratio of squalene:alpha tocopherol is equal to or lessthan 1 as this provides a more stable emulsion. In some cases, theformulation can also contain a stabilizer.

When a combination immunogenic composition with a RSV F proteinpolypeptide antigen is formulated for administration to an infant, thedosage of adjuvant is determined to be effective and relativelynon-reactogenic in an infant subject. Generally, the dosage of adjuvantin an infant formulation is lower (for example, the dose may be afraction of the dose provided in a formulation to be administered toadults) than that used in formulations designed for administration toadult (e.g., adults aged 65 or older). For example, the amount of 3D-MPLis typically in the range of 1 μg-200 μg, such as 10-100 μg, or 10 μg-50μg per dose. An infant dose is typically at the lower end of this range,e.g., from about 1 μg to about 50 μg, such as from about 2 μg, or about5 μg, or about 10 μg, to about 25 μg, or to about 50 μg. Typically,where QS21 is used in the formulation, the ranges are comparable (andaccording to the ratios indicated above). In the case of an oil andwater emulsion (e.g., an oil-in-water emulsion), the dose of adjuvantprovided to a child or infant can be a fraction of the dose administeredto an adult subject.

An immunogenic combination as disclosed herein, or for use in thedisclosed vaccination regimens, methods, uses and kits, typicallycontains an immunologically effective amount (or a fractional dosethereof) of the immunogenic components (and/or polypeptides or nucleicacids) and can be prepared by conventional techniques.

An “immunologically effective amount” is a quantity of a composition(typically, an immunogenic composition) used to elicit an immuneresponse in a subject to the composition or to an antigen in thecomposition. Commonly, the desired result is the production of anantigen (e.g., pathogen)-specific immune response that is capable of orcontributes to protecting the subject against the pathogen. However, toobtain a protective immune response against a pathogen can requiremultiple administrations of the immunogenic composition. Thus, in thecontext of this disclosure, the term immunologically effective amountencompasses a fractional dose that contributes in combination withprevious or subsequent administrations to attaining a protective immuneresponse.

Preparation of immunogenic compositions, including those foradministration to human subjects, is generally described inPharmaceutical Biotechnology, Vol. 61 Vaccine Design—the subunit andadjuvant approach, edited by Powell and Newman, Plenum Press, 1995. NewTrends and Developments in Vaccines, edited by Voller et al., UniversityPark Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation withinliposomes is described, for example, by Fullerton, U.S. Pat. No.4,235,877. Conjugation of proteins to macromolecules is disclosed, forexample, by Likhite, U.S. Pat. No. 4,372,945 and by Armor et al., U.S.Pat. No. 4,474,757.

Typically, the amount of antigen (e.g. protein) or of nucleic acidencoding antigen in each dose of the immunogenic composition is selectedas an amount which induces a protective (or immunoprotective) responsewithout significant, adverse side effects in the typical subject.Protective in this context does not necessarily mean completelyprotective against infection; it means protection against symptoms ordisease, especially severe disease associated with the pathogens. Theamount of antigen can vary depending upon which specific antigen (ornucleic acid) is employed.

Thus, the antigen or nucleic acid that encodes an antigen isadministered at an immunologically effective dose. It will be understoodby those of skill in the art that the immunologically effective amountcan differ between subjects based on parameters such as weight, age, andimmunological and/or physiological status, such that, for example, aninfant dose is generally lower than an adult dose, and a human dose canbe different from the dose administered to an experimental (non-humananimal) subject. For example, a human dose is typically 10×-20× that ofthe dose administered to a mouse. Generally, with respect to thepolypeptide antigen component, it is expected that each human dose willcomprise 1-1000 μg of each protein or antigen, such as from about 1 μgto about 100 μg, for example, from about 1 μg to about 50 μg, such asabout 1 μg, about 2 μg, about 5 μg, about 10 μg, about 15 μg, about 20μg, about 25 μg, about 30 μg, about 40 μg, or about 50 μg.Alternatively, the polypeptide component can be administered in anamount that is between 50 μg and 250 μg, such as about 50 μg, 75 μg, 100μg, 120 μg, 150 μg, 175 μg, 200 μg or 250 μg. These amounts will beunderstood to be illustrative, and an integer or interval within theabove ranges is acceptable.

With respect to the nucleic acid component, the amount is similarlycalculated to provide an immunologically effective amount to the subject(in one or more administrations). Such an amount may be in the case of anucleic acid, between 1 ng and 100 mg. For example, a suitable amount ofa DNA can be from 1 μg to 100 mg. In the case of RNA, a suitable amountcan be from 1 ng to 100 μg. An appropriate amount of the particularnucleic acid (e.g., vector) can readily be determined by those of skillin the art. Exemplary effective amounts of a nucleic acid component canbe between 1 ng and 100 μg, such as between 1 ng and 1 μg (e.g., 100ng-1 μg), or between 1 μg and 100 μg, such as 10 ng, 50 ng, 100 ng, 150ng, 200 ng, 250 ng, 500 ng, 750 ng, or 1 μg (or any integer encompassedwithin or interval inclusive of these amounts). Effective amounts of anucleic acid can also include from 1 μg to 500 μg, such as between 1 μgand 200 μg, such as between 10 and 100 μg, for example 1 μg, 2 μg, 5 μg,10 μg, 20 μg, 50 μg, 75 μg, 100 μg, 150 μg, or 200 μg, or an integer orinterval or fraction between 1 and 200 μg. Alternatively, an exemplaryeffective amount of a nucleic acid can be between 100 μg and 1 mg, suchas from 100 μg to 500 μg, for example, 100 μg, 150 μg, 200 μg, 250 μg,300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg or 1 mg, or anyinteger or interval between 1 μg and 1 mg.

In the case of a recombinant viral vector (e.g., adenovirus) containingthe nucleic acid component is typically administered at a dose that is1×10⁵ to 1×10¹⁵ viral particles, such as from 1×10⁸ to 1×10¹² (e.g.,1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 2.5×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹,1×10¹² particles). Alternatively, a viral vector can be administered ata dose that is typically from 1×10⁵ to 1×10¹⁰ plaque forming units(PFU), such as 1×10⁵ PFU, 5×10⁵ PFU, 1×10⁶ PFU, 5×10⁶ PFU, 1×10⁷ PFU,5×10⁷ PFU, 1×10⁸ PFU, 5×10⁸ PFU, 1×10⁹ PFU, 5×10⁹ PFU, or 1×10¹⁰ PFU. Asabove, any integer or interval within the designated ranges can beadministered.

Generally a human dose will be in a volume of between 0.5 ml and 2 ml.Thus the composition for the uses and methods described herein can beformulated in a volume of, for example 0.5, 1.0, 1.5 or 2.0 ml humandose per individual or combined immunogenic components.

The amount utilized in an immunogenic composition is selected based onthe subject population. An optimal amount for a particular compositioncan be ascertained by standard studies involving observation of antibodytitres and other responses in subjects. Following an initialvaccination, subjects can receive a second administration (e.g., boost)in about 4-12 weeks. For example, when administering an immunogeniccomposition to an infant subject, the initial and subsequentinoculations can be administered to coincide with other vaccinesadministered during this period.

Additional formulation details can be found in WO2010/149745, which isincorporated herein by reference for the purpose of providing additionaldetails concerning formulation of immunogenic compositions comprisingRSV F protein antigens such as PreF analogs.

The immunization embodiment described herein is carried out via asuitable route for administration, such as a parenteral method,including intramuscular, transdermal, intradermal, or cutaneousadministration. For example, the immunization can be carried outcutaneously, which means that the antigen is introduced into the dermisand/or epidermis of the skin (e.g., intradermally). In certain favorableembodiments, the two immunogenic components of the immunogeniccombination are administered colocationally, at or at approximately thesame site on the subject, for example, to the same side or extremity. Inthe case of parenteral administration, colocationally means in proximityat the same (or approximately the same) site on the body, such as to thesame site (e.g., by the same device), or within about 10 cm, or morecommonly within about 5 cm, such as within about 2 cm, or within 1 cm.In some instances, the two or more components are combined(co-formulated) in a single composition for administration to the samesite. Thus, it will be understood that in one favorable embodiment, theadministration of the immunogenic components to a bilaterallysymmetrical subject (such as a human), can be to the co-lateral side ofthe body. That is, the immunogenic component containing the polypeptideantigen and the immunogenic component that contains the nucleic acidthat encodes an antigen are co-laterally administered. Optionally, thetwo components are co-formulated in a single immunogenic compositioneither during manufacture or prior to administration.

Delivery via the cutaneous route including the intradermal route canallow a lower dose of antigen than other routes such as intramusculardelivery. Therefore also provided is an immunogenic combination forcutaneous or intradermal delivery comprising antigens of a respiratorypathogen in a low dose e.g. less than the normal intramuscular dose,e.g. 50% or less of the normal intramuscular dose as provided above forthe protein or nucleic acid components. Optionally the immunogeniccomposition for cutaneous or intradermal delivery also comprises anadjuvant e.g. an metallic salt or QS21 or 3D-MPL or a combinationthereof.

Devices for cutaneous administration include short needle devices (whichhave a needle between about 1 and about 2 mm in length) such as thosedescribed in U.S. Pat. Nos. 4,886,499, 5,190,521, 5,328,483, 5,527,288,4,270,537, 5,015,235, 5,141,496, 5,417,662 and EP1092444. Cutaneousvaccines may also be administered by devices which limit the effectivepenetration length of a needle into the skin, such as those described inWO99/34850, incorporated herein by reference, and functional equivalentsthereof. Also suitable are jet injection devices which deliver liquidvaccines to the dermis via a liquid jet injector or via a needle whichpierces the stratum corneum and produces a jet which reaches the dermis.Jet injection devices are described for example in U.S. Pat. Nos.5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189,5,704,911, 5,383,851, 5,893,397, 5,466,220, 5,339,163, 5,312,335,5,503,627, 5,064,413, 5,520,639, 4,596,556 54,790,824, 4,941,880,4,940,460, WO 97/37705 and WO 97/13537.

Devices for cutaneous administration also include ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis. Additionally, conventional syringes may be used in the classicalmantoux method of cutaneous administration. However, the use ofconventional syringes requires highly skilled operators and thus deviceswhich are capable of accurate delivery without a highly skilled user arepreferred. Additional devices for cutaneous administration includepatches comprising immunogenic compositions as described herein. Acutaneous delivery patch will generally comprise a backing plate whichincludes a solid substrate (e.g. occlusive or nonocclusive surgicaldressing). Such patches deliver the immunogenic composition to thedermis or epidermis via microprojections which pierce the stratumcorneum. Microprojections are generally between 10Dm and 2 mm, forexample 20Dm to 500Dm, 30Dm to 1 mm, 100 to 200, 200 to 300, 300 to 400,400 to 500, 500 to 600, 600 to 700, 700, 800, 800 to 900, 100Dm to400Dm, in particular between about 200Dm and 300Dm or between about150Dm and 250Dm. Cutaneous delivery patches generally comprise aplurality of microprojections for example between 2 and 5000microneedles for example between 1000 and 2000 microneedles. Themicroprojections may be of any shape suitable for piercing the stratumcorneum, epidermis and/or dermis Microprojections may be shaped asdisclosed in WO2000/074765 and WO2000/074766 for example. Themicroprojections may have an aspect ratio of at least 3:1 (height todiameter at base), at least about 2:1, or at least about 1:1. Onesuitable shape for the microprojections is a cone with a polygonalbottom, for example hexagonal or rhombus-shaped. Other possiblemicroprojection shapes are shown, for example, in U.S. Published PatentApp. 2004/0087992. In a particular embodiment, microprojections have ashape which becomes thicker towards the base. The number ofmicroprotrusions in the array is typically at least about 100, at leastabout 500, at least about 1000, at least about 1400, at least about1600, or at least about 2000. The area density of microprotrusions,given their small size, may not be particularly high, but for examplethe number of microprotrusions per cm2 may be at least about 50, atleast about 250, at least about 500, at least about 750, at least about1000, or at least about 1500. In one embodiment of the disclosure thecombination immunogenic composition is delivered to the subject within 5hours of placing the patch on the skin of the host, for example, within4 hours, 3 hours, 2 hours, 1 hour or 30 minutes. In a particularembodiment, the combination immunogenic composition is delivered within20 minutes of placing the patch on the skin, for example within 30seconds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or19 minutes.

The microprojections can be made of any suitable material known to theskilled person. In a particular embodiment at least part of themicroprojections are biodegradable, in particular the tip of themicroprojection or the outer most layer of the microprojection. In aparticular embodiment substantially all the microprojection isbiodegradable. The term “biodegradable” as used herein means degradableunder expected conditions of in vivo use (e.g. insertion into skin),irrespective of the mechanism of biodegradation. Exemplary mechanisms ofbiodegradation include disintegration, dispersion, dissolution, erosion,hydrolysis, and enzymatic degradation.

Examples of microprojections comprising antigens are disclosed inWO2008/130587 and WO2009/048607. Methods of manufacture of metabolisablemicroneedles are disclosed in WO2008/130587 and WO2010/124255. Coatingof microprojections with antigen can be performed by any method known tothe skilled person for example by the methods disclosed in WO06/055844,WO06/055799.

Suitable delivery devices for cutaneous delivery including intradermaldelivery, in the methods and uses described herein include the BDSoluvia™ device which is a microneedle device for intradermaladministration, the Corium MicroCor™ patch delivery system, the GeorgiaTech microneedle vaccine patch, the Nanopass microneedle delivery deviceand the Debiotech Nanoject™ microneedle device. Also provided is acutaneous or intradermal delivery device containing a combinationimmunogenic component or combination as described herein, optionallyformulated with an adjuvant.

The immunogenic combinations can be administered via a mucosal route,including routes, such as intranasal, or oral, that directly place theantigens in contact with the mucosa of the upper respiratory tract.

Thus, the immunogenic combinations, and the components thereof, arecontemplated for use in medicine, and in particular for the preventionor treatment in a human subject of infection by, or disease associatedwith a respiratory pathogen, (such as RSV).

In a particular embodiment of such methods and uses, the subject is ahuman subject. Said human subject may be selected from the group of: aneonate; an infant; a child; an adolescent; an adult; and an elderlyadult. The subject may be a pregnant female with a gestational infant.Alternatively, the subject may not be a pregnant female. Where thesubject is a neonate, administration of the combination immunogeniccomposition may take place within 1 day, or within 1 week, or within 1month of birth.

In connection with the disclosed method for eliciting an immune responseagainst a respiratory pathogen, comprising administering to a subject animmunologically effective amount of the immunogenic combinationdisclosed herein, the elicited immune response against the respiratorypathogen (e.g., RSV) advantageously comprises a protective immuneresponse that reduces or prevents incidence, or reduces severity, ofinfection with the pathogen (e.g., RSV) and/or reduces or preventsincidence, or reduces severity, of a pathological response followinginfection with the pathogen. Said elicited immune response may be abooster response.

Favorably, such administration reduces the symptoms or disease (forexample, pneumonia and/or respiratory distress and failure, or the needfor hospitalization due to severe respiratory disease) in such a cohortby at least about 50%, or at least about 60%, or by 60 to 70%, or by atleast about 70%, or by at least about 80%, or by at least about 90%compared to unvaccinated subjects. Whether there is considered to be aneed for hospitalization due to severe LRTI, or whether a particularcase of LRTI is hospitalized, may vary from country to country andtherefore severe LRTI as judged according to defined clinical symptomswell known in the art may be a better measure than the need forhospitalization.

When the immunogenic combination is administered to an infant, thecomposition can be administered one or more times. The firstadministration can be at or near the time of birth (e.g., on the day ofor the day following birth), or within 1 week of birth or within about 2weeks of birth. Alternatively, the first administration can be at about4 weeks after birth, about 6 weeks after birth, about 2 months afterbirth, about 3 months after birth, about 4 months after birth, or later,such as about 6 months after birth, about 9 months after birth, or about12 months after birth.

As mentioned above, the immunogenic components of the combination foruse in the disclosed vaccination regimens, methods and uses may beco-formulated compositions as described herein, or may be differentcompositions which separately provide each component. Such “separate”compositions may be provided as kits.

In such a kit, the polypeptide antigen of the first immunogeniccomponent and/or the nucleic acid that encodes an antigen of the secondimmunogenic component (as disclosed above) can be contained in one(combined or co-formulated) container or more than one container, suchas in at least one (or one or more) pre-filled syringe. Such a syringemay be a multi-chamber (e.g., dual-chamber) syringe. In the case of amulti-chamber syringe, in an embodiment, the first immunogenic componentis contained within one chamber, and the second immunogenic component iscontained within a second chamber. Prior to administration, the twocomponents can be admixed and then introduced to the subject at the samesite (e.g., through a single needle). In another embodiment, the kitcontains an alternative delivery device, such as a patch as disclosedherein.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

EXAMPLES Example 1: Combined Immunization of CD1 Mice with an AdenoviralVector Expressing RSV F, N and M2.1 Proteins and an AdjuvantedRecombinant F Protein Induces a Broader Immune Response than EachIndividual Vaccine Regimen

Immunogenicity of two doses of the combined PanAd3 RSV (a PanAd 3 vectorcontaining a nucleic acid insert which encodes the amino acid sequencerepresented in SEQ ID NO; 4) and recombinant F (rF) protein/AS04 wasevaluated in mice. Groups of CD1 mice (n=10/group) were immunizedintra-muscularly twice at a 4-week interval with the followingformulations (1^(st) administration/2^(nd) administration). In thisexample, the recombinant F protein was selected to be a conformationallyconstrained F protein analog that was engineered to be stabilized in theprefusion conformation (referred to hereafter in the Examples as rF oras PreF—this is the antigen represented in SEQ ID NO:2). In group 5,adenovirus and recombinant protein were co-administered by 2 injectionsat the same site separated by approximately 10 min.

1^(st) Administration 2^(nd) Administration 1 PanAd3 RSV IM 10⁸ vpPanAd3 RSV IM 10⁸ vp 2 PanAd3 RSV IN 10⁸ vp MVA IM 10⁷ pfu 3 PanAd3 RSVIM 10⁸ vp rF protein 0.5 ug-AS04 4 rF protein-0.5 ug AS04 rF protein 0.5ug-AS04 5 PanAd3 RSV IM 10⁸ vp + PanAd3 RSV IM 10⁸ vp + rF protein 0.5ug-AS04 rF protein 0.5 ug-AS04

Sera from all mice were individually collected on Day 49 (20 days afterthe second immunization) and tested for the presence of RSV neutralizingantibodies using a plaque reduction assay.

Briefly, serial dilutions of each serum were pre-incubated with RSV A(Long strain) at 37° C. After incubation, the virus-serum mixture wastransferred to plates previously seeded with Vero cells. On each plate,cells in one column were incubated with virus only (100% infectivity)and 2 wells received no virus or serum (cell controls). Plates wereincubated for 2 hours at 33° C., medium was removed and RSV mediumcontaining 0.5% CMC (low viscosity carboxymethylcellulose) was added toall wells. The plates were incubated for 3 days at 33° C. beforeimmunofluorescence staining.

For staining, cell monolayers were washed with PBS and fixed with 1%paraformaldehyde. RSV-positive cells were detected using a commercialgoat anti-RSV antiserum followed by a rabbit anti-goat IgG conjugated toFITC. The number of stained plaques per well was counted using anautomated imaging system. Neutralizing antibody titer of each serum wasdetermined as the inverse of the serum dilution causing 60% reduction inthe number of plaques as compared to the control without serum (ED60).Results are illustrated in FIG. 1 . The statistical method employed tocompare different groups was an Analysis of Variance (ANOVA 1) on thelog 10 values.

The cellular response was evaluated by measuring IFNγ-producingsplenocytes 3 weeks after the second immunization. Antigen-specific IFNγproduction by splenocytes was determined by a standard ELISpot assay.Briefly, multiscreen 96-well filtration plates were coated withanti-mouse IFNγ antibody and incubated overnight at +4° C. The followingday, lymphocytes were prepared and incubated in Ag-coated wells for 16hours at 37° C. in the presence of the peptides spanning thecorresponding antigens. After overnight incubation, the cells wereremoved and biotinylated anti-mouse IFNγ was added and incubated 3 hoursat room temperature. For development, alkaline phosphatase-conjugatedstreptavidin was added, followed by the addition of 1-Step NBT-BCIPDevelopment Solution. Plates were acquired and analyzed by an automatedplate reader. ELISpot data were expressed as IFNγ spot forming cells(SFC) per million splenocytes. Results are illustrated in FIG. 2 .

Results presented in FIG. 1 indicate that the co-administration ofPanAd3 RSV and adjuvanted recombinant F protein (co-ad group) inducedthe highest levels of neutralizing antibodies. Additionally, as comparedto two doses of adjuvanted recombinant F protein, the co-ad groupinduced a much higher cellular response (FIG. 2 ). A mirror situationwas observed when comparing the co-ad group to two doses of PanAd3 RSV:the co-ad group induced a slightly higher T-cell response butsignificantly higher neutralizing antibody titers. Although, the primePanAd3 RSV/boost MVA group induced a high cellular response (FIG. 2 ),the neutralizing antibody titers in this group were significantly lowerthan those observed in the co-ad group (FIG. 1 ). In conclusion,co-administration of PanAd3 RSV and adjuvanted recombinant F proteinresulted in the highest combined humoral and cellular responses of allthe tested vaccine regimens.

Example 2: Combined Immunization of Balb/c Mice with an AdenoviralVector Expressing RSV F, N and M2.1 Proteins and an AdjuvantedRecombinant F Protein Induces High Levels of Neutralizing Antibodies andCD8 T-Cell Responses

The immunogenicity of the combined PanAd3 RSV+rF/AS04 was evaluated bymeasuring the neutralizing antibody response as well as identifyingM2.1-specific CD8 T cells in the blood of immunized mice (inbredBalb/c), 14 days after the second immunization. Groups of Balb/c mice(n=11/group) were immunized intra-muscularly twice at a 3-week intervalwith the following formulations (1st/2nd).

Group 1^(st) Administration 2^(nd) Administration 1 PanAd3 RSV IM 10⁸ vpPanAd3 RSV IM 10⁸ vp 2 PanAd3 RSV IM 10⁸ vp + PanAd3 RSV IM 10⁸ vp + rFrF protein 0.5 ug protein 0.5 ug (coformulated) 3 PanAd3 RSV IM 10⁸ vp +PanAd3 RSV IM 10⁸ vp + rF rF protein 0.5 μg-Alum protein 0.5 μg-Alum(colocalized) 4 PanAd3 RSV IM 10⁸ vp + PanAd3 RSV IM 10⁸ vp + rF rFprotein 0.5 μg-AS04 protein 0.5 μg-AS04 0.5 ug (colocalized) 5 PBS PBS

The neutralizing antibody response was evaluated as described in Example1, results are illustrated in FIG. 3 . Two doses of a combinationvaccine composed of PanAd3 RSV and the recombinant F protein (PreF)adjuvanted with either Alum or AS04 induced significantly higher titersthan 2 doses of PanAd3 RSV.

The CD8 T-cell response was measured by identifying M2.1-specific cellsin whole blood with a fluorochrome-tagged pentameric majorhistocompatibility complex (MHC) class I carrying the M2.1 82-90epitope. To this end, blood was collected from each mouse, red bloodcells were lysed, and cells were stained with a fluorescent viabilitymarker, CD3, CD8 and B-220 antibodies and the MHC pentamer. Stainedcells were analyzed by flow cytometry (LSR, Beckton Dickinson) and theproportion of pentamer-positive CD8 T-cells was determined (FIG. 4 ).

Vaccination with two doses of PanAd3 RSV or with any of theco-administration regimens induced a strong M2.1-specific CD8 response.When combined with the neutralizing antibody data, the CD8 T-cell dataindicate that a vaccine regimen consisting of co-administration ofPanAd3 RSV and rF adjuvanted with either Alum or AS04 combines stronghumoral and cellular immune responses.

Example 3: Concurrent Immunization of Balb/c Mice with an AdenoviralVector Expressing RSV F, N and M2.1 Proteins and an AdjuvantedRecombinant F Protein Induces Strong Humoral Immune Responses andProtects from RSV Challenge

Immunogenicity of two doses of co-administered PanAd3 RSV+RSV-rF/AS04was evaluated in Balb/c mice. Groups of Balb/c mice (n=13/group) wereimmunized intra-muscularly twice at a 3-week interval with the followingformulations:

Group 1^(st) Administration 2^(nd) Administration 1 PanAd3 RSV IN 10⁸ vpMVA IM 10⁷ pfu 2 PanAd3 RSV IM 10⁸ vp MVA IM 10⁷ pfu 3 PanAd3 RSV IN 10⁸vp IF protein 2 μg-AS04 4 PanAd3 RSV IM 10⁸ vp IF protein 2 μg-AS04 5PanAd3 RSV IM 10⁸ vp + PanAd3 RSV IM 10⁸ vp + rF protein 2 μg-AS04 rFprotein 2 μg-AS04 6 rF protein 2 μg-AS04 rF protein 2 μg-AS04 7 Live RSV8.3 × 105⁷ pfu no vaccine 8 FI-RSV 1/150 FI-RSV 1/150 9 PBS PBS

In group 5, adenovirus and recombinant protein were co-administered by 2injections at the same site separated by approximately 10 minutes.

Serum was collected 14 days after the second immunization (study day35), at which time animals were challenged intranasally with 2.9×10⁶ pfulive RSV A Long. Lungs from 5 animals were collected 4 days afterchallenge for evaluation of lung viral load. The neutralizing antibodyresponse was evaluated as described in Example 1, 14 days after thesecond immunization.

As observed in CD1 mice, in Balb/c the co-administration of PanAd3 RSVand adjuvanted F (PreF) protein resulted in levels of RSV neutralizingantibodies significantly higher than in all other tested groups, notablytwo doses of adjuvanted F protein or the sequential administration of acombination of PanAd3 RSV and MVA or PanAd3 RSV and recombinant protein(FIG. 5 ).

To measure the efficacy of these exemplary vaccines, lungs wereharvested 4 days post RSV challenge and individually weighed andhomogenized. Serial dilutions (8 replicates each) of each lunghomogenate were incubated with Vero cells and wells containing plaqueswere identified by immunofluorescence, 6 days after seeding. The viraltiter was determined using the Spearman-Kärber method for TCID50calculation and was expressed per gram of lung. Viral replication wasinhibited in the lungs of all vaccinated animals, indicating that alltested vaccine regimens were protective in this model (FIG. 6 ).

Example 4: Concurrent Immunization of Balb/c Mice with an AdenoviralVector Expressing RSV Proteins and an Adjuvanted Recombinant F (PreF)Protein Induces a Th1 Phenotype in Lung T Cells after RSV Challenge anddoes not Induce Lung Eosinophilia or Mucus Production

The effect of vaccination with a regimen comprising an adenovirus vectorRSV candidate and adjuvanted protein on the Th1/Th2 response of lung CD4T-cells, lung eosinophilia and mucus production after challenge wasevaluated in Balb/c mice. FI-RSV was used as a positive control forenhanced pathology. Groups of Balb/c mice (n=12 or 13/group) wereimmunized intra-muscularly twice at a 3-week interval with the followingformulations:

Group 1^(st) Administration 2^(nd) Administration 1 PanAd3 RSV IN 10⁸ vpMVA IM 10⁷ pfu 2 PanAd3 RSV IM 10⁸ vp MVA IM 10⁷ pfu 3 PanAd3 RSV IN 10⁸vp rF protein 2 μg-AS04 4 PanAd3 RSV IM 10⁸ vp rF protein 2 μg-AS04 5PanAd3 RSV IM 10⁸ vp + PanAd3 RSV IM 10⁸ vp + rF protein 2 μg-AS04 rFprotein 2 μg-AS04 6 rF protein 2 μg-AS04 rF protein 2 μg-AS04 7 Live RSV8.3 × 105⁷ pfu no vaccine 8 FI-RSV 1/150 FI-RSV 1/150 9 PBS PBS

In group 5, adenovirus and recombinant protein were co-administered by 2injections at the same site separated by approximately 10 minutes.

Fourteen days after the second immunization (study day 35), animals werechallenged intranasally with 1-3×10⁶ pfu live RSV A Long. Lungs werecollected from 12 animals/group and 4 pools of 3 lungs were prepared.The lungs were minced and incubated in RPMI containing Liberase TL andDNAse for 45 min at 37° C. on orbital shaker. All tissues were thenhomogenized, filtered through sterile 100 μm Nylon cell strainer andlymphocytes were isolated by Percoll gradient. White cells werecollected from the interface and incubated with overlapping peptidesfrom the F antigen for 6 h at 37° C., with addition of Brefeldin A afterthe first 30 min of incubation. Plates were stored overnight at 4° C. Onthe next days, cells were centrifuged, resuspensed, washed, incubatedwith a viability marker, washed, fixed and permeabilized and stainedwith fluorochrome-conjugated antibodies to CD4, CD8, CD45, IL-13 andIFNγ. Cells were acquired on flow cytometer (LSR, Becton Dickinson) andthe percentage of CD45^(pos)CD4^(pos)SCD8^(neg)IFNγ^(pos)/IL-13^(neg)cells (Th1) and CD45^(pos)CD4^(pos)CD8^(neg)IFNγ^(neg)/IL-13^(pos) cells(Th2) cells were determined. The ratio of Th2/Th1 cells was calculated(FIG. 7 ).

FIG. 7 shows that the combination vaccine PanAd3 RSV+rF/AS04 shifts theTh2/Th1 ratio towards Th1 when compared to the rF/AS04 formulation (thebalanced Th2/Th1 ratio of 1 is indicated by the dotted line). All thevaccine formulations containing rF, PanAd3 RSV or MVA combinationsinduce a much lower Th2/Th1 ratio than the one observed in the FI-RSVgroup, a vaccine regimen known to induce high levels of CD4 Th2 cells.

For histopathology, the left lung from 13 animals was collected,inflated in formalin and periodic acid-Schiff staining was performed onformalin-fixed paraffin-embedded (FFPE) mouse lung tissue sections. Thestained slides underwent quantitative analysis using image analysissoftware. For quantitative analysis of PAS-positive tissue(mucus-producing cells), the area (divided by a factor of 10) ofPAS-positive segmented tissue was normalized by the perimeter of theairway epithelium, and expressed as the mean PAS load per millimeter ofbasement membrane (BM)±standard deviation. This was typically performedon 20 airways per subject and the average PAS/mmBM per subject wascalculated (FIG. 8 ).

FIG. 8 shows that the combination vaccine PanAd3 RSV+rF/AS04 is able toreduce the number of mucus-producing cells when compared to the fF/AS04formulation. In addition, the co-administration formulation and allother tested PanAd3 RSV-, MVA- and fF/AS04 combinations inducesignificantly lower mucus-producing cells than the FI-RSV vaccine afterRSV challenge.

Bronchoalveolar lavage (BAL) fluid was collected from the right lunglobe of 8 animals. BAL differential was performed by staining of BALcells with fluorochrome-conjugated antibodies to CD45, CD11c andSiglecF. Cells were acquired on flow cytometer (LSR, Becton Dickinson)and the percentage of CD45^(pos)SiglecF^(pos)CD11C^(neg) (eosinophils)cells was determined. The percentage of eosinophils in BAL was used as amarker of enhanced pathology (FIG. 9 ).

FIG. 9 shows that very low levels of eosinophils are observed in thePanAd3 RSV+fF/AS04 co-administration groups as well as in the othervaccine groups with the exception of FI-RSV. Taken together, the datashown in FIGS. 7 to 9 indicate that the concurrent administrationvaccine composed of PanAd3 RSV and fF/AS04 is not associated withenhanced pathology upon RSV challenge, as shown by a Th1-skewed lung CD4T cell response and low levels of mucus-producing cells and eosinophilsin the lungs.

Example 5: Concurrent Immunization of Balb/c Mice with an AdenoviralVector Expressing RSV Proteins and Adjuvanted Recombinant F Protein atTwo Protein and Three Adjuvant Doses Induces a Neutralizing Antibody anda T Cell Response, and a Th1 Phenotype in Lung T Cells after RSVChallenge, with Significantly Reduced Lung Viral Load

The immunogenicity and effect of vaccination on the Th1/Th2 response oflung CD4 T-cells, lung eosinophilia and mucus production after challengewas evaluated in Balb/c mice with a co-administration regimen comprisingan adenovirus vector (Chimpanzee Adenovirus 155; ChAd155-RSV) RSVcandidate containing a nucleic acid expressing RSV F, N and M2.1 (SEQ IDNO: 4) and adjuvanted protein (PreF, SEQ ID NO:2), at two protein andthree alum adjuvant doses. Groups of Balb/c mice (n=15/group) wereimmunized intra-muscularly, twice at a 3-week interval, with thefollowing formulations concurrently (a few minutes apart):

Group Co-administered: Formulation 1 + Formulation 2 1 ChAd155-RSV IM10⁸ vp + PreF protein 2 μg and Alum hydroxide (50 μg) 2 ChAd155-RSV IM10⁸ vp + PreF protein 2 μg and Alum hydroxide (17 μg) 3 ChAd155-RSV IM10⁸ vp + PreF protein 2 μg and Alum hydroxide (6 μg) 4 ChAd155-RSV IM10⁸ vp + PreF protein 0.2 μg and Alum hydroxide (50 μg) 5 ChAd155-RSV IM10⁸ vp + PreF protein 0.2 μg and Alum hydroxide (17 μg) 6 ChAd155-RSV IM10⁸ vp + PreF protein 0.2 μg and Alum hydroxide (6 μg) 7 ChAd155-RSV RSVIM 10⁸ vp + PreF protein 2 μg and AS04D (50 μg) 8 PBS + ChAd155-RSV RSVIM 10⁸ vp 9 PreF protein 2 μg-and Alum hydroxide (50 μg) + PBS 10 AlumAdsorbed FI-RSV 1/150 11 Live RSV~3.6 × 10⁶ pfu 12 PBS + PBS

Immunogenicity: Sera from 11 mice/group were individually collected onDay 35 (14 days after the second immunization) and tested for thepresence of RSV neutralizing antibodies using a plaque reduction assayas described in Example 1. Results are illustrated in FIG. 10 . Twodoses of a combination vaccine composed of concurrently administeredChAd155-RSV and recombinant F (PreF) protein adjuvanted with either Alumor AS04 induced similar neutralizing antibody titres than 2 doses ofprotein adjuvanted with 50 μg of Alum, but significantly higher titersthan 2 doses of ChAd155-RSV. The combination of ChAd155-RSV andadjuvanted protein allowed reduction of the dose of Alum to 17 μg whilemaintaining a neutralizing antibody response similar to that of theprotein adjuvanted with 50 μg (alone or concurrently with ChAd155-RSV).A dose response was observed between the 2 μg and 0.2 μg protein dosesand the lowest level of Alum (6 μg) resulted in lower titres ofneutralizing antibody at both protein doses.

The CD8 T-cell response was measured by identifying M2.1-specific cellsin whole blood with a fluorochrome-tagged pentameric majorhistocompatibility complex (MHC) class I carrying the M2.1 82-90epitope. To this end, blood was collected from 5 mice/group, 14 daysafter the second immunization. Red blood cells were lysed, and cellswere stained with a fluorescent viability marker, CD3, CD8 and B-220antibodies and the MfHC pentamer. Stained cells were analyzed by flowcytometry (LSR, Beckton Dickinson) and the proportion ofpentamer-positive CD8 T-cells was determined (FIG. 11 ). A similar CD8response was detected in whole blood T calls in all groups immunizedwith ChAd155 RSV, but not in the group immunized with PreF proteinalone.

Response to Challenge: Mice were challenged intranasally with 1-3×10⁶pfu live RSV A Long. At day 4 post-challenge, lungs were harvested andindividually weighed and homogenized. Serial dilutions (8 replicateseach) of each lung homogenate were incubated with Vero cells and wellscontaining RSV plaques were identified by immunofluorescence, 6 daysafter seeding. The viral titer was determined using the Spearman-Kärbermethod for TCID50 calculation and was expressed per gram of lung (FIG.12 ). Most animals in the ChAd155-RSV and/or adjuvanted protein groupsshowed full protection (undetectable viral load in lungs). There was atrend for slightly lower protection with the lowest alum dose.

Lungs were collected from 4 animals/group at 4 days post-challenge andprepared as described in Example 4. Following restimulation oflymphocytes with pooled peptides from the F antigen, cells were stainedfor flow cytometry analysis of intracellular cytokines as described inExample 4 and the ratio of Th2/Th1 cells was calculated (FIG. 13 ).Following restimulation of lung lymphocytes with pooled peptides fromthe M2.1 antigen carried out in an analogous fashion, the proportion ofIFNγ-expressing CD8+ T cells was calculated (FIG. 14 ).

FIG. 13 shows that ChAd155 RSV+PreF (at all adjuvant doses) shifts theTh2/Th1 ratio towards Th1 when compared to the PreF/Alum formulation(the balanced Th2/Th1 ratio of 1 is indicated by the dotted line) andthe choice and dose of adjuvant has little impact. All the vaccineformulations containing ChAd155 RSV+PreF combinations also induce alower Th2/Th1 ratio than that observed in the FI-RSV group, a vaccineregimen known to induce high levels of CD4 Th2 cells and associated withenhanced RSV disease.

FIG. 14 shows that high levels of INFg-expressing CD8+ T cells weredetected in the lungs of all of the co-administration ChAd155 RSV+PreFgroups. At the higher protein dose, reduction in Alum correlated withhigher CD8 response, but the adjuvant level had little impact at thelower protein dose. CD8 levels were lower in the lungs of mice immunizedwith ChAd155 RSV alone than in the co-administration groups, andundetectable in the PreF protein/Alum group.

For histopathology, Periodic Acid Schiff staining was performed toquantify mucus-producing cells on formalin-fixed paraffin-embedded(FFPE) mouse lung tissue sections as described in Example 4.Mucus-producing cells are associated with enhanced RSV pathology inducedby FI-RSV in the Balb/c RSV challenge model. The average PAS/mmBM persubject was calculated (FIG. 15 ).

FIG. 15 shows that the co-administration of ChAd155-RSV with PreF/Alumdoes not increase the number of mucus-producing cells after RSVchallenge when compared to the PreF/Alum formulation. At the higherprotein dose, reduction in Alum correlated with lower numbers ofmucus-producing cells, but the adjuvant level had less impact at thelower protein dose. The combination of ChAd155-RSV with PreF/AS04Dinduced lower levels of mucus-producing cells than the equivalentChAd155-RSV/PreF+Alum combination. The lowest number of mucus-producingcells was seen with ChAd155-RSV administered alone.

The results in FIGS. 10-15 indicate that the concurrent administrationvaccine composed of ChAd155 RSV and adjuvanted PreF protein antigen isable to induce protective neutralizing antibody and T cell responseswith a Th1/Th2 balanced CD4 response and no enhanced pathology upon RSVchallenge.

SEQUENCE LISTINGSEQ ID NO: 1: Nucleotide sequence of an exemplary conformationally constrainedPreF antigen.ATGGAGCTGCTGATCCTGAAAACCAACGCCATCACCGCCATCCTGGCCGCCGTGACCCTGTGCTTCGCCTCCTCCCAGAACATCACCGAGGAGTTCTACCAGTCCACCTGCTCCGCCGTGTCCAAGGGCTACCTGTCCGCCCTGCGGACCGGCTGGTACACCTCCGTGATCACCATCGAGCTGTCCAACATCAAGGAAAACAAGTGCAACGGCACCGACGCCAAGGTGAAGCTGATCAAGCAGGAGCTGGACAAGTACAAGAGCGCCGTGACCGAACTCCAGCTGCTGATGCAGTCCACCCCTGCCACCAACAACAAGTTTCTGGGCTTCCTGCAGGGCGTGGGCTCCGCCATCGCCTCCGGCATCGCCGTGAGCAAGGTGCTGCACCTGGAGGGCGAGGTGAACAAGATCAAGAGCGCCCTGCTGTCCACCAACAAGGCCGTGGTGTCCCTGTCCAACGGCGTGTCCGTGCTGACCTCCAAGGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCCTATCGTGAACAAGCAGTCCTGCTCCATCTCCAACATCGAGACCGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAGATCACCCGCGAGTTCTCCGTGAACGCCGGCGTGACCACCCCTGTGTCCACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTGATCAACGACATGCCTATCACCAACGACCAGAAAAAACTGATGTCCAACAACGTGCAGATCGTGCGGCAGCAGTCCTACAGCATCATGAGCATCATCAAGGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCTCTGTACGGCGTGATCGACACCCCTTGCTGGAAGCTGCACACCTCCCCCCTGTGCACCACCAACACCAAGGAGGGCTCCAACATCTGCCTGACCCGGACCGACCGGGGCTGGTACTGCGACAACGCCGGCTCCGTGTCCTTCTTCCCTCTGGCCGAGACCTGCAAGGTGCAGTCCAACCGGGTGTTCTGCGACACCATGAACTCCCTGACCCTGCCTTCCGAGGTGAACCTGTGCAACATCGACATCTTCAACCCCAAGTACGACTGCAAGATCATGACCAGCAAGACCGACGTGTCCTCCAGCGTGATCACCTCCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCTCCAACAAGAACCGGGGAATCATCAAGACCTTCTCCAACGGCTGCGACTACGTGTCCAATAAGGGCGTGGACACCGTGTCCGTGGGCAACACACTGTACTACGTGAATAAGCAGGAGGGCAAGAGCCTGTACGTGAAGGGCGAGCCTATCATCAACTTCTACGACCCTCTGGTGTTCCCTTCCGACGAGTTCGACGCCTCCATCAGCCAGGTGAACGAGAAGATCAACGGGACCCTGGCCTTCATCCGGAAGTCCGACGAGAAGCTGCATAACGTGGAGGACAAGATCGAGGAGATCCTGTCCAAAATCTACCACATCGAGAACGAGATCGCCCGGATCAAGAAGCTGATCGGCGAGGCCSEQ ID NO: 2: Amino acid sequence of an exemplary conformationally constrainedPreF antigen.MELLILKTNAITAILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKSAVTELQLLMQSTPATNNKFLGFLQGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVOLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPLAETCKVOSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINGTLAFIRKSDEKLHNVEDKIEEILSKIYHIENEIARIKKLIGEASEQ ID NO: 3: Nucleotide sequence of an exemplary nucleic acid that encodes RSVantigens.ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCCGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGAGCAAGGGCTACCTGAGCGCCCTGAGAACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAAGAAAACAAGTGCAACGGCACCGACGCCAAAGTGAAGCTGATCAAGCAGGAACTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACCGGGCCAGACGGGAGCTGCCCCGGTTCATGAACTACACCCTGAACAACGCCAAAAAGACCAACGTGACCCTGAGCAAGAAGCGGAAGCGGCGGTTCCTGGGCTTTCTGCTGGGCGTGGGCAGCGCCATTGCCAGCGGCGTGGCCGTGTCTAAGGTGCTGCACCTGGAAGGCGAAGTGAACAAGATCAAGAGCGCCCTGCTGAGCACCAACAAGGCCGTGGTGTCCCTGAGCAACGGCGTGAGCGTGCTGACCAGCAAGGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCAGCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGGGAGTTCAGCGTGAACGCCGGCGTGACCACCCCTGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGTCCATCATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACCAACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGACAGAGGCTGGTACTGCGACAACGCCGGCAGCGTGTCATTCTTTCCACAGGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCTCCGAAGTGAACCTGTGCAACGTGGACATCTTCAACCCCAAGTACGACTGCAAGATCATGACCTCCAAGACCGACGTGTCCAGCTCCGTGATCACCTCCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAGGGGGTGGACACCGTGTCCGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACGAGTTCGACGCCAGCATCAGCCAGGTGAACGAGAAGATCAACCAGAGCCTGGCCTTCATCCGGAAGTCCGACGAGCTGCTGCACAATGTGAATGCCGGCAAGTCCACCACCAACCGGAAGCGGAGAGCCCCTGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAATCCCGGCCCTATGGCCCTGAGCAAAGTGAAACTGAACGATACACTGAACAAGGACCAGCTGCTGTCCAGCAGCAAGTACACCATCCAGCGGAGCACCGGCGACAGCATCGATACCCCCAACTACGACGTGCAGAAGCACATCAACAAGCTGTGCGGCATGCTGCTGATCACAGAGGACGCCAACCACAAGTTCACCGGCCTGATCGGCATGCTGTACGCCATGAGCCGGCTGGGCCGGGAGGACACCATCAAGATCCTGCGGGACGCCGGCTACCACGTGAAGGCCAATGGCGTGGACGTGACCACACACCGGCAGGACATCAACGGCAAAGAAATGAAGTTCGAGGTGCTGACCCTGGCCAGCCTGACCACCGAGATCCAGATCAATATCGAGATCGAGAGCCGGAAGTCCTACAAGAAAATGCTGAAAGAAATGGGCGAGGTGGCCCCCGAGTACAGACACGACAGCCCCGACTGCGGCATGATCATCCTGTGTATCGCCGCCCTGGTGATCACAAAGCTGGCCGCTGGCGACAGATCTGGCCTGACAGCCGTGATCAGACGGGCCAACAATGTGCTGAAGAACGAGATGAAGCGGTACAAGGGCCTGCTGCCCAAGGACATTGCCAACAGCTTCTACGAGGTGTTCGAGAAGTACCCCCACTTCATCGACGTGTTCGTGCACTTCGGCATTGCCCAGAGCAGCACCAGAGGCGGCTCCAGAGTGGAGGGCATCTTCGCCGGCCTGTTCATGAACGCCTACGGCGCTGGCCAGGTGATGCTGAGATGGGGCGTGCTGGCCAAGAGCGTGAAGAACATCATGCTGGGCCACGCCAGCGTGCAGGCCGAGATGGAACAGGTGGTGGAGGTGTACGAGTACGCCCAGAAGCTGGGCGGAGAGGCCGGCTTCTACCACATCCTGAACAACCCTAAGGCCTCCCTGCTGTCCCTGACCCAGTTCCCCCACTTCTCCAGCGTGGTGCTGGGAAATGCCGCCGGACTGGGCATCATGGGCGAGTACCGGGGCACCCCCAGAAACCAGGACCTGTACGACGCCGCCAAGGCCTACGCCGAGCAGCTGAAAGAAAACGGCGTGATCAACTACAGCGTGCTGGACCTGACCGCTGAGGAACTGGAAGCCATCAAGCACCAGCTGAACCCCAAGGACAACGACGTGGAGCTGGGAGGCGGAGGATCTGGCGGCGGAGGCATGAGCAGACGGAACCCCTGCAAGTTCGAGATCCGGGGCCACTGCCTGAACGGCAAGCGGTGCCACTTCAGCCACAACTACTTCGAGTGGCCCCCTCATGCTCTGCTGGTGCGGCAGAACTTCATGCTGAACCGGATCCTGAAGTCCATGGACAAGAGCATCGACACCCTGAGCGAGATCAGCGGAGCCGCCGAGCTGGACAGAACCGAGGAATATGCCCTGGGCGTGGTGGGAGTGCTGGAAAGCTACATCGGCTCCATCAACAACATCACAAAGCAGAGCGCCTGCGTGGCCATGAGCAAGCTGCTGACAGAGCTGAACAGCGACGACATCAAGAAGCTGAGGGACAACGAGGAACTGAACAGCCCCAAGATCCGGGTGTACAACACCGTGATCAGCTACATTGAGAGCAACCGCAAGAACAACAAGCAGACCATCCATCTGCTGAAGCGGCTGCCCGCCGACGTGCTGAAAAAGACCATCAAGAACACCCTGGACATCCACAAGTCCATCACCATCAACAATCCCAAAGAAAGCACCGTGTCTGACACCAACGATCACGCCAAGAACAACGACACCACCSEQ ID NO: 4: Amino acid sequence of an exemplary nucleic acid that encodes RSVantigens.MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPREMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVOLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVOSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNRKRRAPVKQTLNEDLLKLAGDVESNPGPMALSKVKLNDTLNKDQLLSSSKYTIQRSTGDSIDTPNYDVQKHINKLCGMLLITEDANHKFTGLIGMLYAMSRLGREDTIKILRDAGYHVKANGVDVTTHRQDINGKEMKFEVLTLASLTTEIQINIEIESRKSYKKMLKEMGEVAPEYRHDSPDCGMIILCIAALVITKLAAGDRSGLTAVIRRANNVLKNEMKRYKGLLPKDIANSFYEVFEKYPHFIDVFVHFGIAQSSTRGGSRVEGIFAGLFMNAYGAGQVMLRWGVLAKSVKNIMLGHASVQAEMEQVVEVYEYAQKLGGEAGFYHILNNPKASLLSLTQFPHESSVVLGNAAGLGIMGEYRGTPRNQDLYDAAKAYAEQLKENGVINYSVLDLTAEELEAIKHQLNPKDNDVELGGGGSGGGGMSRRNPCKFEIRGHCLNGKRCHFSHNYFEWPPHALLVRONFMLNRILKSMDKSIDTLSEISGAAELDRTEEYALGVVGVLESYIGSINNITKOSACVAMSKLLTELNSDDIKKLRDNEELNSPKIRVYNTVISYIESNRKNNKQTIHLLKRLPADVLKKTIKNTLDIHKSITINNPKESTVSDTNDHAKNNDTTPositions 1-524 = FΔTM protein Positions 525-552 = 2a sequence Positions553-943 = N protein Positions 944-1146 = M2-1 protein

1-34. (canceled)
 35. An immunogenic combination of compositionscomprising: a) a first immunogenic composition comprising an F proteinantigen of respiratory syncytial virus (RSV) constrained in thepre-fusion conformation; and b) a second immunogenic compositioncomprising an adenoviral vector comprising a nucleic acid encoding an Fprotein antigen of respiratory syncytial virus (RSV); wherein the firstimmunogenic composition and the second immunogenic composition areformulated in a single composition (co-formulated).
 36. The immunogeniccombination of claim 35, wherein the antigens of the first and secondimmunogenic compositions comprise one or more identical immunogenicepitopes.
 37. The immunogenic combination of claim 35, wherein the firstand at least second immunogenic composition comprise a plurality ofantigens.
 38. The immunogenic combination of claim 35, wherein thesecond immunogenic composition comprises a nucleic acid that encodes anectodomain of an RSV F Protein (FΔTM).
 39. The immunogenic combinationof claim 35, wherein the second immunogenic composition comprises anucleic acid that encodes an RSV FΔTM antigen and RSV M2-1 and Nantigens.
 40. The immunogenic combination of claim 39 wherein aself-cleavage site is included between the RSV FΔTM antigen and the RSVM2-1 and a flexible linker is included between the RSV M2-1 and Nantigens.
 41. The immunogenic combination of claim 35, wherein the atleast one of the first and/or second immunogenic composition furthercomprises one or more selected from the group consisting of: a carrier,an excipient, a buffer and an adjuvant.
 42. The immunogenic combinationof claim 41, wherein the adjuvant comprises one or more selected fromthe group consisting of: a metallic salt, 3-D-monophosphoryl-lipid-A(MPL), a saponin, and an oil and water emulsion, a liposome and ananoparticle.
 43. The immunogenic combination of claim 42, wherein themetallic salt is (i) an aluminum salt selected from the group consistingof: aluminum hydroxide, aluminum potassium sulfate, aluminumhydroxyphosphate sulfate, and aluminum phosphate, or (ii) a calcium saltis selected from the group consisting of: calcium phosphate and calciumfluoride.
 44. A method for eliciting an immune response specific forrespiratory syncytial virus (RSV) in a subject, the method comprising astep of administering the immunogenic combination of claim 35 to thesubject.
 45. A method for preventing, reducing, or treating an infectionby respiratory syncytial virus (RSV) in a subject comprising a step ofadministering the immunogenic combination of claim 35 to the subject.46. A process for making the immunogenic combination of claim 35comprising a step of co-formulating a first immunogenic compositioncomprising an F protein antigen of respiratory syncytial virus (RSV)constrained in the pre-fusion conformation and a second immunogeniccomposition comprising an adenoviral vector comprising a nucleic acidencoding an F protein antigen of respiratory syncytial virus (RSV). 47.A method of vaccination for the prevention, reduction or treatment ofinfection by respiratory syncytial virus (RSV), comprising a step ofconcurrently administering an immunogenic combination of claim 35.